Interferon antagonists useful for the treatment of interferon related diseases

ABSTRACT

The present invention relates to a process for ameliorating or preventing diseases that are caused, in part, by an increased level of, and/or an abnormal responsivity to, interferon. Specifically, the invention provides compositions and methods for preventing and treating subjects suffering from, or at risk for, such diseases. Such methods include the administration of a pharmacological preparation of interferon binding proteins that antagonize interferon&#39;s action. This invention comprises compositions of interferon binding proteins that can inhibit the activity of Type I and II.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/284,740, filed Oct. 31, 2002, now U.S. Pat. No. 7,285,526, issuedOct. 23, 2007, which is a continuation-in-part of U.S. application Ser.No. 09/845,260, filed Apr. 30, 2001, now abandoned, which is acontinuation of U.S. application Ser. No. 09/067,398, filed Apr. 28,1998, now abandoned, which is a continuation of U.S. application Ser.No. 08/502,519, filed Jul. 14, 1995, now U.S. Pat. No. 5,780,027, issuedJul. 14, 1998. The entire teaching of the above applications areincorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates to a process for ameliorating orpreventing diseases that are caused, in part, by an increased level of,and/or an abnormal responsivity to, interferon. Alzheimer's disease, HIVinfection, Down syndrome, transplant rejection, autoimmune disease, andinfant encephalitis are examples of such diseases. Specifically, theinvention provides a method for treating subjects suffering from, or atrisk for, such diseases by the administration of a pharmacologicalpreparation of interferon binding proteins of mammalian and/or viralorigin that antagonize interferon's action. This invention comprisescompositions of interferon binding proteins that can inhibit theactivity of interferon gamma plus interferon alpha such compositionsalong with their method of production and modification being describedherein.

2. Background of the Invention

The Molecular Biology of Interferons and Interferon Receptors

Interferons are proteins that alter and regulate the transcription ofgenes within a cell by binding to interferon receptors on the regulatedcell's surface and thus prevent viral replication within the cells.There are five types of interferons (IFN), which are designated α(formerly α1), ω (formerly α2), β, γ and τ. Mature human interferons arebetween 165 and 172 amino acids in length. In humans IFN-α and IFN-ω areencoded by multiple, closely related non-allelic genes. Additionally,there are pseudo-genes of IFN-α and IFN-ω. By contrast, IFN-β and IFN-γare encoded by unique genes.

The interferons can be grouped into two types. IFN-γ is the sole type IIinterferon; all others are type I interferons. Type I and type IIinterferons differ in gene structure (type II interferon genes havethree exons, type I one), chromosome location (in humans, type II islocated on chromosome-12; the type I interferon genes are linked and onchromosome-9), and the types of tissues where they are produced (type Iinterferons are synthesized ubiquitously, type II by lymphocytes). TypeI interferons competitively inhibit each others binding to cellularreceptors, while type II interferon has a distinct receptor. Reviewed bySen, G. C. & Lengyel, P., 1992, J. Biol. Chem. 267:5017-5020.

Although all type I interferons compete for binding to a common receptoror receptors, the effects of different type I interferons can bedifferent. Pontzer, C. H., 1994, J. Interfer. Res. 14:133-41.Additionally, there appears to be several kinds of type I interferonreceptor. For example, there is evidence that the type I interferonreceptors of different cell types are different. Benoit, P., 1993, J.Immunol. 150:707. The number of genes encoding the type I interferonreceptors is unknown: however, the genes appear to be linked to eachother and to at least one gene encoding an IFN-γ receptor component aswell. In humans, chromosome region 21q21.1-21.31 encodes all the genesneeded for the receptor for type I interferon (Raziuddin, A., 1984,Proc. Natl. Acad. Sci. 81:5504-08; Soh, J., 1993, Proc. Natl. Acad. Sci.90:8737-41; Soh, J., 1994, J. Biol. Chem. 269:18102-10) and at least oneessential component of the type II interferon receptor (Jung, V., 1990,J. Biol. Chem. 265:1827-30).

It is becoming increasingly clear that the interferons have an importantrole in neuromodulation (Hori, T. et al., 1998, Neuroimmunomodulation5:172-177; Dafny, N., 1998, Brain Res. Brain Res. Rev. 26:1-15) andneurodegeneracy (Blasko, I. et al., 2001, J. Neuroimmunol. 116:1-4). Ofparticular relevance is the observation of neuropathology associatedwith the overproduction of either IFN-α (Akwa, Y. et al., 1998, J.Immunol. 161:5016-5026) or IFN-γ (Corbin, J. G. et al., 1996, Mol CellNeurosci. 7:354-370) in the transgenic mouse brain. Evidence has beenpresented in support of a role for IFN-α in neuromodulation (reviewed byDafney (Dafny, N., 1998, Brain Res Brain Res Rev. 26:1-15)).

The potential role of IFN-γ in neurodegeneracy has only recently beenrecognized (Blasko, I. et al., 2001, J Neuroimmunol. 116:1-4). Evidencehas been presented for a direct role for IFN-γ in neuron apoptosis ofneurons in culture (Hallam, D. M. et al., 1998, Neurosci. Lett.252:17-20) through the induction of neuron caspase activity (Hallam, D.M. et al., 2000, J. Neuroimmunol. 110:66-75).

The Biology of Interferon Action and Down Syndrome

The binding of interferons to their receptor, leads to a cascadepost-translational modification to other proteins which are thentransported to the nucleus where they regulate the transcription ofgenes by binding to specific nucleic acid sequences. The nucleic acidsequence which is characteristic of genes responsive to type Iinterferons is designated the Interferon Sensitive Response Element(ISRE). Reviewed Tanaka, T. & Taniguchi, T., 1992, Adv. Immunol. 52:263.Type-I interferons are synthesized in response to viral infection,except for IFN-τ which is constitutively produced in the placenta; TypeII interferons are synthesized in response to antigen stimulation.

Interferons alter the rates of synthesis and the steady state levels ofmany cellular proteins. An overall effect of interferon is usually aninhibition of cellular proliferation.

The possibility that cells from subjects having Down syndrome may haveabnormal responsivity to interferon was introduced by the discovery thata gene encoding an interferon inducible protein, which was subsequentlyidentified as the type I interferon receptor, was located onchromosome-21. Tan Y. H. et al., 1974, J. Exp. Med. 137:317-330. Thisobservation prompted comparisons of the response of diploid andtrisomy-21 aneuploid cultured cells to interferon added to the culturemedium. These studies have consistently shown an increased responsivityof trisomy-21 cells to interferon. Tan, Y. H. et al., 1974, Science186:61-63; Maroun, L. E., 1979, J. Biochem. 179:221; Weil, J. et al.,1983, Hum. Genetics 65:108-111; reviewed Epstein, C. J., & Epstein, L.B., 8 LYMPHOKINES pp 277-301 (Academic Press, New York, 1983); Epstein,C. J. et al., 1987, ONCOLOGY AND IMMUNOLOGY OF DOW SYNDROME (Alan R.Liss, 1987). The publications of these studies have been accompanied byspeculative conjectures that the altered responsivity to interferonplayed a role in the pathogenesis of lesions of Down syndrome. See,Maroun, L. E., 1980, J. Theoret. Biol. 86:603-606.

Down Syndrome and Animal Models of It

An animal model of Down syndrome has been constructed by use of theknowledge that human chromosome-21 is syntenic to mouse chromosome-16,i.e., that many of the genes present on each are homologs of each other.Mice having specified trisomies can be bred by use of parental micehaving “Robertsonian” chromosomes, i.e., chromosomes that areessentially the centromeric fusion of two different murine chromosomes.A variety of such Robertsonian chromosomes have been identified,including at least two involving chromosome-16 and a second differentchromosome: Rb(16.17) and Rb(6.16). Mice homozygous for any Robertsonianor combination of independent Robersonian chromosomes are euploid andfertile.

The intercross (F1) between an Rb(16.17) and an Rb(6.16) mouse is alsofully diploid at each genetic locus, although errors in meiosis maycause reduced fertility. Note that in such an F1 both the maternal andpaternal chromosome-16 are a part of a Robertsonian chromosome.

Because of meiotic errors the outcross between a mouse having both twodifferent Robertsonian chromosome-16's and a non-Robertsonian mousegives rise to a trisomy-16 conceptus in between 15% and 20% of cases.Gearhart, J. D. et al., 1986, Brain Res. Bull. 16:789-801; Gropp, A. etal., 1975, Cytogenet. Cell Genet. 14:42-62. The murine trisomy-16fetuses develop to term but do not live beyond birth by more than a fewhours.

Examination of the fetal trisomy-16 and the postpartum human trisomy-21reveals a number of analogous or parallel lesions. For this reason, themurine trisomy-16 construct is considered to be an animal model of Downsyndrome. Epstein, C. J., THE METABOLIC BASIS OF INHERITED DISEASE, 6THED. pp 291-326 (McGraw-Hill, New York, 1989); Epstein, C. J. et al.,1985, Ann. N.Y. Acad. Sci. 450:157-168. Because a murine trisomy-16fetus is not viable post partum, the opportunity to study theneurological pathology of the model has been limited. However, it isclear that in both human trisomy-21 and murine trisomy-16 there is anoverall reduction in fetal size and particularly in the development ofthe fetal brain. Epstein, C. J., THE CONSEQUENCES OF CHROMOSOMEIMBALANCE: PRINCIPLES, MECHANISMS AND MODELS (Cambridge UniversityPress, New York, 1986). Further insights into the effects of marinetrisomy-16 have been obtained by the formation of Ts16←→2N chimeras(Gearhart, J. D. et al., 1986, Brain Res. Bulletin 16:815-24) and bytransplantation of fetal-derived Ts16 tissue into a 2N host (Holtzman,D. M. et al., 1992, Proc. Natl. Acad. Sci. 89:138387; Holtzman, D. M. etal., DOWN SYNDROME AND ALZHEIMER DISEASE, pp 227-44 (Wiley-Liss, NewYork, 1992).

Alzheimer's Disease and Amyloid Precursor Protein

Alzheimer's disease is a progressive dementia which is characterized bythe precipitation of a peptide, termed an A β peptide, of about 40 aminoacids within the brain and within the walls of blood vessels in thebrain. The A β peptide is derived from the processing of a larger cellsurface protein called the β Amyloid Precursor Protein (β APP).Production of the A β peptide is not per se pathological. The functionsof both the A β peptide or β APP are unknown.

Several lines of evidence indicate that the deposition of the A βpeptide is not merely correlative but rather causative of Alzheimer'sdisease. The gene encoding β APP is located on chromosome-21 and, asnoted above, subjects having Down syndrome develop Alzheimer's disease.More directly, kinship groups have been identified among the many causesof familial Alzheimer's disease in which the inheritance of the Diseaseis linked to the inheritance of a gene encoding a mutated β APP,moreover the mutation is within the A β peptide itself. Reviewed Selkoe,D. J., 1994, Ann. Rev. Neurosci. 17:489-517. Transgenic mice, havingmultiple copies of such a mutant β APP gene, operatively linked to astrong, neuronal and glial cell specific promoter, develop theanatomical lesions of Alzheimer's disease at about 6-9 months of age.Games, D. et al., 1995, Nature 373:523.

There is a relationship between Down syndrome and Alzheimer's disease.The gene encoding the β APP is found on chromosome-21. Patients withDown syndrome are at increased risk of developing Alzheimer's disease orAlzheimer's-like pathology, most often by about the fifth decade of lifealthough cases of earlier development have been reported. Mann, D. M. A.et al., 1990, Acta Neuropathol. 80:318-27.

Aids and Inceased IFN Levels

After a latency period that can last for many years, HIV infectedindividuals “convert” to the immunosuppressed state referred to as“AIDS”. Acquired Immunodeficiency Syndrome (“AIDS”) is a complex ofvarious pathologies that is proceeded by and associated with increasedlevels of IFN-γ and IFN-α in the blood (Rossel, S. et al., 1989, J.Infectious Diseases 159:815-821) and IFN-α in the CSF (Rho, M B. et al.,1995, Brain, Behavior, and Immunity 9:366-77). Immunization with humanIFN-α to reduce IFN levels is associated with the prevention ofconversion to AIDS and improved prognosis for AIDS patients (Gringeri,A. et al., 1996, J AIDS and Human Retrovirology 13:55-67) as taught byZagury, et al. (U.S. Pat. No. 6,093,405). However, this immunizationprocedure has serious limitations as it is both irreversible andunreliable.

Interferons, like cytokines in the body generally, do not act in theabsence of antagonism (Van Weyenbergh, J. et al., 1998, J. Immunol.161:1568-1574.; Paludan, S. R., 1998, Scand. J. Immunol. 48:459-468.;Ghosh, A. K. et al., 2001, J. Biol. Chem. 276:11041-11048) and/orsynergy (Kwon, S. et al., 2001, Nitric Oxide 5:534-546.; Moore, P. E. etal., 2001, J. Appl. Physiol. 91:1467-1474.; Zhang, Y. et al., 2001, J.Interferon Cytokine Res. 21:843-850) caused by other cytokines or otherinterferon types. Note that some other cytokine combinations have beenfound to not be synergistic (Czuprynski, C. J. et al., 1992, Antimicrob.Agents Chemother. 36:68-70). In addition, the action of one type ofinterferon frequently can be mimicked or replaced by the action ofanother type of interferon (Hughes, T. K. et al., 1987, J InterferonRes. 7:603-614). There is speculation that if you inhibit IFN-γ andIFN-α then disease can be treated (Lachgar, A. et al., 1994, BiomedPharmacother. 48:73-77, U.S. Pat. No. 5,780,027), however conflictingdata in the literature suggests that combined treatment may, in someinstances, not be more effective than monotherapy (Lukina, G. V. et al.,1998, Ter. Arkh. 70:32-37).

Presented herein is evidence demonstrating that the pathologicalnegative effects of one type of interferon (IFN-γ) are in the body aidedand enhanced in its negative effects by another type of interferon(IFN-α). These data demonstrate that the reduction of interferonbioactivity to relieve a pathological condition can be measurablyimproved by reducing the activity of both interferon typessimultaneously. See, e.g., FIG. 3, which show the results of single vs.double knockout of interferon receptor genes.

SUMMARY OF THE INVENTION

The present invention is based, in part on the recognition that incertain pathologic processes, the host is rendered hyperproductiveand/or aberrantly sensitive to the effects of interferon so that theeffects of interferon become an immediate and direct cause of thepathology. Such processes include, in humans, trisomy of chromosome-21or the portion of the chromosome-21 that encodes the receptor for type Iinterferon and at least one component of the receptor for IFN-γ, whichis the genetic abnormality associated with Down syndrome. Other diseaseswhere IFN plays an important role include Alzheimer's disease andlate-stage HIV infection.

The present invention provides a method of ameliorating the pathologiceffects of interferon by administering to a subject, in the above-notedcircumstances, an antagonist of interferon. Embodiments of the inventioninclude the administration of antagonists, alone or in combination, thatare antagonists of Type I interferon, Type II interferon (IFN-γ), andplacental interferon (IFN-τ).

The present invention relates to compositions of interferon antagoniststhat inhibit the activity of interferons from either animal and/or humansources such compositions being comprised of proteins, or dimers orfragments thereof, that bind to IFN-γ together with proteins, or dimersor fragments thereof, that bind to IFN-α such compositions thus bindingto and inhibiting both IFN-γ and IFN-α. The present inventionencompasses pharmacologically useful formulations of proteins that bindto and inhibit interferon comprising at least one IFN-γ binding proteinplus at least one protein that binds to and inhibits one or more IFN-αsubtypes that are appropriately modified as needed to provide forincreased serum half-life, reduced immunogenicity, efficient mode ofadministration, increased binding affinity, and/or enhanced blood brainbarrier (BBB) penetration.

The invention features a composition for preventing or decreasing thepathological effects of a disease, where the pathological effects areassociated with an increased level of or a heightened responsiveness tointerferon, where the composition comprises at least two isolatedinterferon binding proteins, and where the composition inhibits theactivity of at least two different species of interferon. The species ofinterferon can comprise IFN-α and IFN-γ. The isolated interferon bindingproteins can include a first interferon binding protein that binds toIFN-α and a second interferon binding protein that binds to IFN-γ. Oneor more of the interferon binding proteins can be an IFN-α bindingprotein. One or more of the interferon binding proteins can be an IFN-γbinding protein. One of the interferon binding proteins can be the humanIFN-γ receptor. The interferon binding proteins can be the B8R and B18Rproteins of Vaccinia virus. One of the interferon binding proteins canbe Vaccinia B18R IFN-α binding protein. One of the interferon bindingproteins can be the Vaccinia R8R interferon-γ binding protein. One ormore of the interferon binding proteins can be an antibody, or anantibody fragment. One or more of the interferon binding proteins can bean interferon-specific antibody. One or more of the interferon bindingproteins can be an interferon-specific humanized antibody. Antibodies orantibody fragments can be PEGylated or fused with transferrin. Theinterferon binding proteins can be PEGylated or fused with transferrin.

In another aspect, the invention features a plurality of interferonbinding proteins comprising two or more interferon binding proteins,where the interferon binding proteins bind to and inhibit the activityof one or more interferons (i.e., interferon species).

Any of the compositions can be used in a method of treating a diseasecaused by elevated levels of two or more interferons, where the methodincludes the step of administering a therapeutically effective amount ofthe composition. The disease can be Alzheimer's disease, Down'ssyndrome, infant encephalitis, or an autoimmune disease. The disease canbe HIV. Administration of the composition can be used to prevent theonset of AIDS.

In a further aspect, the invention features a method of treating adisease, where the method includes the step of administering atherapeutically effective amount of a pair of interferon antagonists toa patient having or at risk of having said disease, where theadministration results in a reduction in the level of bioavailableinterferon, such that the disease in the patient is treated. The diseasecan be Alzheimer's disease, Down's syndrome, infant encephalitis, or anautoimmune disease. The disease can be HIV. Administration of thecomposition can be used to prevent the onset of AIDS. Administration ofthe composition can be used to prevent or ameliorate AIDS-associateddementia. The interferon antagonists can be PEGylated or fused withtransferrin.

In an additional aspect, the invention features a method of treating orpreventing transplant rejection, where the method includes administeringa therapeutically effective amount of one or more interferon antagoniststo a patient, wherein the administration of the interferon antagonistreduces the level of bioavailable interferon in the patient such thatthe transplant rejection is treated or prevented. The interferonantagonists can be PEGylated or fused with transferrin.

The invention also features a method of treating or preventing AIDS inan HIV-infected patient, where the method includes administering atherapeutically effective amount of one or more interferon antagoniststo the HIV infected patient, where the administration of the interferonantagonist reduces the level of bioavailable interferon in the patientsuch that AIDS in the HIV-infected patient is treated or prevented Theinterferon antagonists can be PEGylated or fused with transferrin. Theone or more interferon antagonists can be a homodimer of the human IFN-γreceptor ligand binding subunits, or a heterodimer of the AR1 and AR2ligand binding subunits of the human IFN-α receptor.

In an additional aspect, the invention features a method of treating orpreventing IFN related disease using the B18R gene product or itshomologs of viral or other origin. The B18R gene product or its homologcan be PEGylated, or fused to transferrin.

In a further aspect, the invention features a method of treating orpreventing IFN related disease using the B8R gene product or itshomologs of viral or other origin. The B18R gene product or its homologcan be PEGylated, or fused to transferrin.

The invention also features a composition for preventing or decreasingthe pathological effects of a disease, wherein the pathological effectsare associated with an increased level of or a heightened responsivenessto interferon, wherein said composition comprises one or more isolatedinterferon binding protein, and wherein the composition inhibits theactivity of one or more species of interferon. The interferon bindingprotein can be PEGylated or fused with transferrin. The interferonbinding protein can be PEGylated or fused with transferrin. Theinterferon binding protein can be B18R or B8R.

As used herein, “interferon binding proteins” refers to a protein orprotein fragment or peptide that binds to interferons with a Kd bindingconstant of at least 10-3, preferably 10-5, more preferably 10-7 andmost preferably 10-9 or less and inhibit the biological activity of thetargeted interferon. The binding constants of typical interferon bindingproteins are shown in Table 2, below.

As used herein, the term “interferons” refers to the different types ofinterferons (IFN), (Diaz, M. O. et al 1994, J Interferon Res.14:221-222; Allen, D et al, 1996, J Interferon Res. 16:181-184). Thesefive are designated a interferon (formerly α1), ω interferon (formerlyω2), β interferon, γ interferon, and τ interferon. The sequences of theα, β and γ interferons are shown in FIG. 7.

Interferon-α and -β are so-called type I interferons. They are secretedby a wide variety of cell types and have a wide range of functions. Theyare best known for their antiviral properties. They mediate theireffects through the same receptor, which is present of the surfaces ofvirtually all nucleated cell types. Interferon-gamma (immune or type IIinterferon) is distinct in several ways from both interferon-alpha and-beta. It mediates its effects through a separate receptor from the oneused by the type I interferons. In addition to having antiviralproperties, it is especially noteworthy as a potent modulator of thefunctions of a wide range of cell types. Many of these functions arecritical to the immune and inflammatory responses.

As used herein, INF-α refers to interferon-α subspecies and dimersthereof. INF-β refers to interferon-β subspecies and dimers thereof.

As used herein, to “inhibit the activity” refers to a decrease in theactivity or available amount of an interferon that is at least 10%,preferably 10-30%, more preferably 30-50% and most preferably 50-100%decreased in the presence of one or more interferon binding proteins ascompared to the activity of an interferon in the absence of theinterferon binding proteins.

As used herein, the term “fusion polypeptide” or “fusion protein” refersto a polypeptide that is comprised of two or more amino acid sequences,wherein the two or more amino acid sequences are physically linked by apeptide bond and wherein the two or more amino acid sequences are notfound linked in nature.

As used herein, an “iron transport protein” is a transferrin preferablyselected from the group comprising human transferrin, lactoferrin,ovotransferrin and/or serum transferrin.

As used herein, “Vaccinia B18R IFN-α binding protein” refers to aglycoprotein (60-65 kDa; see FIG. 8 that exists in a soluble and amembrane-bound form. The protein functions as a type-1 interferon (IFN)receptor with broad species specificity. The B18R protein has highaffinity for human IFN-α and also binds rabbit, bovine, rat, pig, andmouse IFN-α. Since the protein exists as a soluble extracellular and acell surface protein it has the potential to block both autocrine andparacrine functions of IFN. The B18R protein has been shown to inhibitthe antiviral potency of IFN-α-1, IFN-α-2, IFN-α-8/1/8, and IFN-Ω onhuman cells.

As used herein, “Vaccinia B8R interferon γ binding protein” refers to aprotein encoded by the B8R open reading frame of vaccinia virus (seeFIG. 8). B8R possesses a hydrophobic amino-terminal signal sequence butlacks a discernible membrane anchor domain, suggesting that the proteinsmay be secreted.

As used herein, “modified to enhance drug delivery” refers tomodifications of interferon antagonists so as to fiacilitate theirdelivery to a target tissue. In one embodiment the invention includespost-translational modification of interferon antagonists (for examplePEGylation or glycation). In a further embodiment the interferonantagonist is fused to a fusion protein. In a preferred embodiment thefusion protein is an iron transport protein that promotes transport ofthe interferon antagonist across the blood brain barrier. In a furtherembodiment, the interferon antagonist is fused to a fusion protein thatis capable of multimerization.

As used herein, a “therapeutically effective amount” means the totalamount of each active component of the pharmaceutical composition ormethod that is sufficient to show a meaningful patient benefit, i.e., inthe treatment, healing, prevention or amelioration of the relevantmedical condition, or an increase in rate of treatment, healing,prevention or amelioration of such conditions. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. Generally, a composition will be administered in asingle dose in the range of 100 μg-10 mg/kg body weight, preferably inthe range of 1 μg-100 μg/kg body weight. This dosage may be repeateddaily, weekly, monthly, yearly, or as considered appropriate by thetreating physician.

As used herein, “interferon antagonist” refers to a biomolecule thatbinds to an interferon as defined herein and blocks the biologicalactivity of the interferon. In a preferred embodiment the interferonantagonist of the invention is an interferon binding protein. Theeffective amount of an interferon antagonist needed to bind and blockinterferon proteins in the blood can be determined by assaying theconcentration of bioavailable interferon. An effective dose ofinterferon antagonist is a dose that is sufficient to reduce the levelof bioavailable interferon by between at least three to five fold, morepreferably by about ten fold and most preferably by about twenty fivefold below the normal levels of interferon. The interferon antagonistcan be a mixture of antagonists that are specific for the variousdifferent types of interferon. When one type of interferon predominates,the antagonist can be an antagonist for only the predominate type ofinterferon that is present.

As used herein, “transplant rejection” refers to the immune responsethat results from the transfer of a donor's cells, tissues or organsinto a histoincompatible host.

As used herein, “treating a disease” refers to arresting or otherwiseameliorating the symptoms of a disease at least 10%, preferably 20-50%and more preferably 75-100%.

As used herein, the term “autoimmune disease” refers to a disorderwherein the immune system of a mammal mounts a humoral or cellularimmune response to the mammal's own tissue or has intrinsicabnormalities in its tissues preventing proper cell survival withoutinflammation,

Examples of autoimmune diseases include, but are not limited to,diabetes, rheumatoid arthritis, multiple sclerosis, lupus erythematosis,myasthenia gravis, scieroderma, Crohn's disease, ulcerative colitis,Hashimoto's disease, Graves' disease, Sjögren's syndrome. polyendocrinefailure, vitiligo, peripheral neuropathy, graft-versus-host disease,autoimmnune polyglandular syndrome type L, acute glomerulonephritis,Addison's disease, adult-onset idiopathic hypoparathyroidism (AOIH),alopecia totalis, amyotrophic lateral sclerosis, ankylosing spondylitis,autoimmune aplastic anemia, autoimmune hemolytic anemia, Behcet'sdisease, Celiac disease, chronic active hepatitis, CREST syndrome,dermatomyositis, dilated cardiomyopathy, eosinophilia-myalgia syndrome,epidermolisis bullosa acquisita (EBA), giant cell arteritis,Goodpasture's syndrome, Guillain-Barré syndrome, hemochromatosis,Henoch-Schönlein purpura, idiopathic IgA nephropathy, insulin-dependentdiabetes mellitus (IDDM), juvenile rheumatoid arthritis, Lambert-Eatonsyndrome, linear IgA dermatosis, myocarditis, narcolepsy, necrotizingvasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome,pemphigoid, pemphigus, polymyositis, primary sclerosing cholangitis,psoriasis, rapidly-progressive glomerulonephritis (RPGN), Reiter'ssyndrome, stiff-man syndrome and thyroiditis.

As used herein, “infant encephalitis” refers to inflammation of thebrain most commonly caused by a viral infection of the brain that occursin young children. The most important viruses causing sporadic cases ofencephalitis in immunocompetent adults are herpes simplex virus type 1(HSV-1), varicella-zoster virus (VZV), and, less commonly,enteroviruses. Epidemics of encephalitis are caused by arboviruses,which belong to several different viral taxonomic groups includingAlphavirus of the family Togaviridae (e.g., Eastern equine encephalitisvirus, Western equine encephalitis virus), Flavivirus of the familyFlaviviridae (e.g., St. Louis encephalitis virus, Powassan virus), andBunyavirus of the family Bunvaviridae (e.g., California encephalitisvirus serogroup, LaCrosse virus).

As used herein, “bioavailable” refers to the portion of interferon thatcan be absorbed, transported, and/or utilized physiologically.

As used herein, the term “immunoglobulin” or “antibody” refers to aconventional antibody molecule, as well as fragments thereof which arealso specifically reactive with one of the subject polypeptides.Antibodies can be fragmented using conventional techniques, e.g.,protein digestion, gene truncation, and the fragments screened forutility in the same manner as described herein for whole antibodies. Forexample, F(ab)2 fragments can be generated by treating antibody withpepsin. The resulting F(ab)2 fragment can be treated to reduce disulfidebridges to produce Fab fragments. The antibodies and antibody fragmentscan be screened for enhanced binding affinity using phage displaytechnology. The antibody of the present invention is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for a polypeptide conferred by at least one CDR regionof the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and able to be detected, (e.g., thelabel can be a radioisotope, fluorescent compound, chemiluminescentcompound, enzyme, or enzyme co-factor). The antibodies, monoclonal orpolyclonal and its hypervariable portion thereof (FAB, FAB2, etc.) aswell as the hybridoma cell producing the antibodies are a further aspectof the present invention which find a specific industrial application inthe field of diagnostics and monitoring of specific diseases, preferablythe ones hereafter described. In a preferred embodiment, theimmunoglobulin is humanized.

“Humanized antibody”, as used herein, refers to antibody molecules inwhich amino acids have been replaced in the non-antigen binding regionsin order to more closely resemble a human antibody, while stillretaining the original binding ability. Methods for making humanizedantibodies are described in U.S. Pat. Nos. 6,054,297, 5,859,205, whichare hereby incorporated be reference in their entirety.

As used herein, “in frame” refers to the reading frame used for thetranslation of a fusion polypeptide nucleotide sequence. In a fusionpolypeptide X-Y, coding sequences for polypeptide Y are said to be ‘inframe’ with upstream coding sequences for the polypeptide X if thetranslation of the coding sequences X-Y results in a fusion polypeptidewherein polypeptide X is fused to polypeptide Y.

As used herein, the “pathological effects are associated with anincreased level of, or a heightened responsiveness to, interferon”refers to a disease state that is associated with elevated levels of IFNreceptors on the patient's cells or bioavailable interferon in thebodily fluids of a patient afflicted with the disease. For example, FIG.3 shows evidence that the pathological negative effects of one type ofinterferon (IFN-γ) in this instance, growth retardation of a trisomy 16mouse fetus is enhanced in its negative effects by another type ofinterferon (IFN-α). These data on growth retardation also demonstratethat reduction of interferon bioactivity through gene knock outmutations in the IFN-γ and/or IFN-α/β receptors results in reduction inthe pathological condition i.e., growth retardation and that thisreduction is enhanced if the activity of both interferon types, i.e.,IFN-γ and IFN-α are reduced simultaneously.

As used herein, the term “Human Immunodeficiency Virus” or “HIV” ismeant to refer to all strains of human immunodeficiency viruses. Activehuman immunodeficiency virus infection results in a decline in thenumber of CD4+ T cells, which in turn results in the incapacity of theinfected individual to mount an effective immune response to viral,bacterial, fungal or parasitic infections.

As used herein, the “AIDS” refers to HIV infected patients with a CD4+ Tcell count of less than <200/μL and who develop one of theHIV-associated diseases considered to be indicative of a severe defectin cell-mediated immunity, typically opportunistic infections byorganisms such as P. carinii, atypical mycobacteria, CMV, fungi, andother organisms that do not ordinarily cause disease in the absence of acompromised immune system.

As used herein, AIDS in an HIV infected patient is said to he “treated”or “prevented” if the CD4+ T cell count remain at or increases to avalue that is 25%, 50%, 75%, 90%, 99% or preferably equal to the CD4+ Tcell count of a patient that is not infected with HIV.

As used herein, the onset of AIDS in an HIV infected patient is said tobe “prevented” if the patient's CD4+ T cell count decreases no more than25%, preferably 10%, most preferably 0% from the CD4+ T cell count of apatient who is not infected with HIV.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. The lengths of Trisomy 16 fetuses plotted as a function ofthe average length of normal littermates. FIG. 1A, Uninjected controls;FIG. 1B, non-specific IgG (ns-IgG) injected controls; FIG. 1C, anti-IFNinjected fetuses. An analysis-of-covariance was performed to compare thegroups on length while adjusting for average normal littermate length.The lengths of the anti-IFN treated group were significantly greaterthan those of the ns-IgG injected controls (p=0.0112) and those of theuninjected controls (p=0.0037). The dotted lines in each figureencompass the 95% confidence limits.

FIGS. 2A-2B. Morphometric analysis of the development in normal, Trisomy16 treated and Trisomy 16 sham treated fetuses; FIG. 2A, average eyeopening of 17 to 23 mm trisomy 16 fetuses; FIG. 2B, average backcurvature scores of trisomy 16 fetuses greater than 20 mm in length.Columns. (A) Uninjected; (B) non-specific IgG injected; (C) anti-IFNinjected; (D) euploid. The mean. +/−-standard error is presented.

FIG. 3: Comparison of the improvement in trisomy 16 mouse fetus growthwith a single (IFN-γ only) vs. double (IFN-γ plus IFN-α/β) receptor geneknockout. To produce the partial interferon receptor knockout trisomy(PIRKOT) mouse fetus, double IFN-γ+ IFN-α/β R−/− knockout males (B & KInternational) were mated to double translocation females [Rb(6,16)24LuBX Rb(16,17)7BNRFI, Jackson Labs]. Crown-to-Rump length was measured onday 15-19 fetuses. Growth retardation is expressed as a percent of meaneuploid littermate length. Knockout of a single IFN-γ receptor gene wassufficient to significantly improve trisomy fetus growth rate (meangrowth retardation: 9.15+/−1-49%, N=9, p=0.043). However, the doubleknockout was measurably more effective (mean growth retardation:6.81+/−1.6%, N=8, p=0.008).

FIG. 4: Steps in the Cloning of Vaccinia IFN Inhibitor Genes. FIG. 4A:PCR amplification of the B18R Vaccinia gene (agarose gelelectrophoresis). Lane 1: Lambda DNA HindIII digest markers; Lane 2:B18R gene PCR product (expected length, 999 bp). FIG. 4B: Restrictionenzyme digests of the mammalian expression plasmid carrying the B18RVaccinia gene (agarose gel electrophoresis). Lane 1: HindIII digest ofthe plasmid (there are no HindIII sites in the insert and there is oneHindIII site in the original plasmid); Lane 2: BgIII plasmid digest(there is one BgIII site in the insert and one BgIII site in theoriginal plasmid); and Lane 3: Lambda DNA HindIII digest markers.

FIG. 5: Truncated human interferon γ receptor gene isolation. The humanIFN-γ receptor gene is, by example, here isolated by PCR amplificationfrom a thymus cDNA library (Clontech, Palo Alto, Calif., USA) for PCRsubcloning into expression plasmids using the following primers:P1:ATGGCTCTCCTCTTTCTCCTA (SEQ ID NO: 1), P2:TCTAGAACCTTTTATACTGCTATTGAA(SEQ ID NO: 2. Lane 1: Truncated IFN-γ receptor gene. Lane 2: Lambda DNAmarkers.

FIG. 6A: Example of a modified homodimer of the human IFN-γ receptor.

FIG. 6B. Example of a modified heterodimer with one copy each of bothα-interferon receptor subunits (AR1 and AR2). The hinge region providesfor two characteristics: (1) a flexible link to preventreceptor-Transferrin (Tf) mutual interference; and, (2) a signal toinstruct the protein synthesis machinery of the eukaryotic cell to linktwo polypeptides together. The Tf provides the fusion protein with alonger serum half:life and the ability to be actively transported intothe brain via the Tf receptors found lining the walls of the bloodvessels of the brain.

FIGS. 7A, 7B, 7C: Genbank Accession numbers of the interferons (SEQ IDNOS 9-18 respectively, in order of appearance).

FIGS. 8A and 8B: Nucleotide and amino acid sequence of B18R and B8R geneproducts of Vaccinia virus (B8R, Genbank Accession No.: AFO162273; B18R(FIG. 8A), GenBank Accession No.: D90076 (FIG. 8B)) (SEQ ID NOS 19-22respectively, in order of appearance).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of ameliorating the pathologiceffects of interferon by administering to a subject, in the above-notedcircumstances, an antagonist of interferon. Embodiments of the inventioninclude the administration of antagonists, alone or in combination, thatare antagonists of Type I interferon, Type II interferon (IFN-γ), andplacental interferon (IFN-τ).

Many different species of virus produce interferon-binding proteins as ameans of reducing the ability of the host to mount an immune response toinfection, Examples of interferon binding proteins of the invention aredepicted in Table 1, below, and share the property of being able to bindIFN-α or IFN-γ with high affinity, as shown in Table 2.

TABLE 2 Examples of binding constants of interferon binding proteins. KdReference Gamma-IFN: Myxoma M-T7 1.2 × 10⁻⁹ M Bai, H. et al., 2002,Biotechniques 32: 160, 162-4, 166-71. Vaccinia B8R Undetermined Humangamma 1-2 × 10⁻⁹ M Fountoulakis, M. et al., 1990, J. Biol. receptorChem. 265: 13268-75 Alpha-IFN: Vaccinia B18R 174 × 10⁻¹² M Symons, J. A.et al., 1995, Cell 81: 551-60 Human alpha 1 × 10⁻⁷ M Nguyen, N. Y. etal., 1996, J receptor (in solution) Interferon Cytokine Res 16 835-44 1× 10⁻⁹ M (cell surface) Immunoglobin 10⁻¹¹ M Darsley, M. J. et al.,1985, Embo J. (Ig) 10⁻⁷ (Ag) to 4: 383-92; Klein, B. et al., 1995, 10⁻⁸M (peptide) Immunol Today 16: 216-20

In order to ensure a successful infection, viruses use many differentstrategies to suppress or circumvent the host immune response. Forexample, the CrmA gene encoded by poxvirus functions by inhibitinginterleukin-1 beta converting enzyme (Pickup, D. J., Infect. Agents Dis.3(2-3):116-127 (1994), the SERP-1 gene of Myxoma virus encodes a serineprotease inhibitor (McFadden, G. et al., J. Leukoc. Biol. 57(5):731-738(1995)) and, the Myxoma virus encodes a TNF receptor homologue (T2, U.S.Pat. No. 5,464,938).

“Cytokine binding proteins” belong to a group of virally coded proteinstermed “viroceptors” (Upton et al., Virology, 184:370 (1991)) as theyact as decoy receptors to bind to cytokines thereby diverting thecytokine away from its normal host cell surface receptor. The term“virally encoded interferon binding proteins” refers to a viroceptorthat binds to and inhibits one or more interferon types.

The Myxoma virus also encodes an IFN binding protein M-T7 that isdescribed in U.S. Pat. No. 5,834,319. However, the M-T7 protein is oflimited utility as it does not bind to interferon of human origin (see,e.g., U.S. Pat. No. 5,834,419). In contrast, the B8R and B18R proteinsencoded by the poxviruses which are the subject of the present invention(FIG. 8) bind to and strongly inhibit the activity of human IFN-γ (B8R)and human IFN-α (B18R).

In some instances it may be preferable to use an IFN binding protein ofhuman origin. The present invention describes highly modified forms ofthe human IFN-γ and IFN-α/β receptors that provide for increased bindingaffinity, serum half-life, and enhanced blood-brain-barrier (BBB)penetration.

Selection of Subjects

The present invention concerns the administration of interferonantagonists to subjects in order to ameliorate the neurological,pathological, and developmental abnormalities in the subject due to theaction of interferon. A particular group of subjects at risk aresubjects having a trisomy of the portion of the chromosome region,designated in humans 21q21.1-21.31, that encodes for interferonreceptors. This group has the clinical diagnosis of Down syndrome.Grete, N., 1993, Eur. J. Hum, Genetics 1:51-63; Sinet, P. M., 1994,Biomed. & Pharmacol. 48:247-252. The homologous chromosome in mice ischromosome-16.

Diagnosis of Down syndrome can be made by any method known to themedical arts. Typically, for diagnosis in utero, amniocentesis can beperformed at about 14 weeks of gestational age and chorionic villussampling (biopsy) can be performed between 9 and 12 weeks of gestationalage. Down syndrome in children and adults is diagnosed from karyotypesof peripheral blood cells. Cells from either type of sample are culturedand cytogenetic examination can be performed by methods well understoodby those skilled in the art.

As noted above, patients having Down syndrome are at increased risk todevelop Alzheimer's disease. A further group of subjects that wouldbenefit from the invention consist of subjects having the diagnosis ofprobable Alzheimer's disease or who are at increased risk of developingAlzheimer's disease from causes other than Down syndrome. The diagnosisof probable Alzheimer's disease is made by clinical criteria (McKhann,G., 1984, Neurology 34:939; DIAGNOSTIC AND STATISTICAL MANUAL OF MENTALDISORDERS IV, American Psychological Association, Washington, D.C.).Persons having a familial predisposition to Alzheimer's disease are alsosuitable subjects for the present invention.

HIV Infected Pre-AIDS and AIDS Patients

A further group of patients that would benefit from this inventionconsist of subjects having the diagnosis of HIV infection who havedeveloped, or are at risk of developing, the AIDS complex of pathologiesassociated with immunosuppression. In these individuals, the conversionto the AIDS complex is proceeded by, and associated with, increasedIFN-γ and IFN-α bioactivity which can be determined by various methodswell known to the medical arts.

The Selection of Antagonists

The antagonist of the invention can be any antagonist that can beadministered to the subject in an amount effective to prevent thedeleterious action of the interferon.

The effective amount of antagonists that act by binding to and blockinginterferon proteins in the blood can be determined by assaying theconcentration of bioavailable interferon in the subjects blood. Aneffective dose of antagonist is a dose that is sufficient to reduce thelevel of bioavailable interferon by between at least three to five fold,more preferably by about ten fold and most preferably by about twentyfive fold below the normal levels of interferon.

The assay of bioavailable interferon is performed by adding a sample ofthe subjects blood to a culture of an interferon sensitive cell linewhich is then infected with a test virus, typically Vesicular StomatitisVirus (VSV), and the number of viral plaques is determined or thecytotoxic effects of the VSV infection is otherwise quantitated.Bioavailable interferon blocks productive viral infection. The level ofbioavailable interferon is calculated by comparing various dilutions ofthe test sample with a titration of a standard sample of interferon.Such assays are routine in the art. See, e.g., Hahn, T. et al., 1980, inINTERFERON: PROPERTIES AND CLINICAL USES, ed. by A. Khan, N. O. Hill andG. L. Dorn, (Leland Fikes Foundation Press, Dallas, Tex.); Armstrong, J.A., 1971, Applied Microbiology 21:723-725; Havell, E. A. & Vilcek, J.1972, Anti-microbial Agents and Chemotherapy 2:476-484.

In one embodiment of the invention the antagonist is a monoclonalanti-interferon antibody or fragment thereof. The production of suchantibodies is well known in the art. The production of anti-IFN-αmonoclonal antibodies that block interferon activity is taught by U.S.Pat. No. 4,973,556 to Bove et al. The production of blocking monoclonalantibodies to IFN-γ is taught by U.S. Pat. No. 4,948,738 to Banchereau.The structure of human trophoblastic interferon (IFN-τ) has beenrecently disclosed (Whaley, A. E., 1994, J. Biol. Chem. 269:10864-8).Monoclonal antibodies and other antagonists to this interferon can beproduced using methods well know to those skilled in the art.

In a preferred embodiment, the antibody is a “chimeric” antibody, i.e.,an antibody having a variable region from one species and a constantregion from another species. Most typically chimeric antibodies for usein humans have constant regions of human origin. In an alternativepreferred embodiment, the antibody is a “grafted” antibody, i.e., anantibody having complementarity determining regions from one species anda constant region and a framework region of the variable region from asecond species. A grafted antibody in which the second species is humanis termed a “humanized” antibody. Methods of making chimeric antibodiessuitable for pharmaceutical use are disclosed in International Patentpublication WO 92/16553 by Le, J. (Oct. 1, 1992). “Grafted” antibodiesand “humanized” antibodies are described in U.S. Pat. No. 5,225,539 toWinter and International Patent publications WO 91/09967 and WO 92/11383by Adair, J. R. et al. Suitable antagonists, smaller than an antibodymolecule, can be derived from anti-interferon monoclonal antibodies bytechniques well known in the art. See, e.g. U.S. Pat. No. 5,091,513 toHuston and U.S. Pat. No. 5,260,203 to Ladner. As used herein the term“antibody antagonists” includes natural polyclonal and monoclonalantibodies, chimeric and grafted antibodies, and antibodies produced byhuman antibody gene trangenic animals, and enzymatically andrecombinantly produced interferon binding fragments of each type ofantibody.

In an alternative embodiment the antagonist can be a recombinantlyproduced protein that comprises the interferon binding portion of aninterferon receptor. The production of soluble interferon receptors bybaculovirus transduced cells is described in Fountoulakis et at., 1991,Eur. J. Biochem. 198:441-450. Alternatively the antagonist can be afusion protein that contains an interferon binding domain of aninterferon receptor.

In alternative embodiments, the antagonist can be an antibody to aninterferon receptor, a soluble interferon receptor, receptor fragment,or a peptide that is derived from an interferon that occupies thereceptor binding site but does not activate the receptor. Such an IFN-γpeptide antagonist is disclosed by Jarpe, M. A. et al., 1993, J.Interferon Res. 13:99-103.

When the subject is a fetus, or an infant less than 6 weeks of age, theblood brain barrier is not fully formed. In these circumstancesantibodies and other proteins that block the interferon receptor candirectly reach the central nervous system. When the subject has anintact blood brain barrier, an embodiment of the invention can employantibodies and proteins that block interferon by binding the interferondirectly, rather than those that act at the interferon receptor.

Alternatively, increased CNS entry of antibody antagonists can beobtained by chemical modification of the antagonist. Such modificationsinclude cationization, Pardridge, W., 1991, “Peptide Drug Delivery tothe Brain”, and glycation, Poduslo, J. F. & Curran, G. L., 1994,Molecular Brain Research 23:157.

The interferon antagonist can be a mixture of antagonists that arespecific for the various different types of interferon. When one type ofinterferon predominates, the antagonist can be an antagonist for onlythe predominate type of interferon that is present.

When the subject is a fetus, then the antagonist can be administered bya transplacental route, e.g., antibody that is transported across theplacenta. The human isotypes IgG1, IgG3 and IgG4 are suitable fortransplacental administration.

Interferon Antagonists

In a preferred embodiment, Interferon binding proteins particularlyuseful for the formulation of the type of interferon antagonistdescribed in the present invention are the B18R and B8R gene products ofVaccinia virus (B8R; B18R, see FIG. 8). The B8R protein is an IFN-γbinding protein that will bind and inhibit IFN-γ from human, rat, orrabbit sources (Alcami, A. et al., 1995, J. Virol. 69:4633-4639). Thecross-species binding ability of these proteins greatly improves theirutility as their ability to bind interferon, and the side effects ofthis binding, can be tested in animals prior to the start of humantrials. In addition, the B18R protein is an IFN-α binding protein thatis a single polypeptide that will bind and inhibit numerous subspeciesof IFN-α from various sources (e.g., human, rat, mouse, or rabbit)(Symons, J. A. et al., 1995, Cell. 81:551-560).

In another embodiment, the invention is comprised of proteins of bothviral and human origin. For example, the human IFN-γ receptor, or dimeror fragment or modification thereof, could be in a composition with theVaccinia B18R protein, or dimer or fragment or modification thereof. Theisolation and preparation of IFN-γ receptors is taught by U.S. Pat. Nos.5,578,707, 5,221,789, and 5,763,210. The human IFN-γ ligand-binding genecan be cloned and expressed using standard procedures. For example, thisgene can be PCR amplified from a thymus cDNA library (Clontech, PaloAlto, Calif., USA) using the procedures and primers described herein(see FIG. 5).

The genes encoding the IFN binding proteins of the invention can beisolated and expressed in prokaryotic or eukaryotic systems usingmethods familiar to those practiced in the art of gene cloning andexpression.

Interferon Antagonist Fusion Proteins

The invention provides for interferon antagonist fusion proteins,fragments, dimiers and modifications thereof. Methods used to link thecDNA nucleic acid encoding a gene of interest, or portion thereof, inframe, to a nucleic acid encoding interferon antagonist are well knownto those in the art and are described extensively in Ausubel et al.,eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,1995, with supplemental updates. The encoded-protein of interest may belinked in-frame to the amino-or carboxyl-terminus of the interferonantagonist. The nucleic acid encoding the chimeric protein is thenlinked in operable association to a promoter element of a suitableexpression vector. Fusion partners are chosen to confer specificproperties to the interferon antagonist such as the ability to dimerizeor multimerize or the ability to traverse the blood-brain barrier.

In a preferred embodiment of the invention, interferon antagonists arefused to the human transferrin protein, e.g., Genbank accession number:XM_(—)002793 which facilitates passage across the blood brain barrier(see “Modifications of Interferon Antagonist Proteins”, infra).

In a preferred embodiment of the invention, interferon antagonists aredimers with an enhanced binding affinity for interferons. For example,human IFN-γ can be a dimer, and its interaction with the homodimer ofIFN-γ ligand-binding subunits provides a significantly stronger bondthan one subunit alone. This improvement in binding affinity has beendemonstrated by Moosmayer and co-workers (Moosmayer, D. et al., 1995, J.Interferon. Cytokine Res. 15:1111-1115). An example of the human IFN-γreceptor fragment modified to form a dimer and further modified toenhance BBB transport via fusion to the human transferrin protein ispresented in FIG. 6A.

Primers According to the Invention

The invention provides for oligonucleotide primers useful for amplifyinginterferon antagonist sequences, such as those cDNA sequences thatencode the Vaccinia B8R or B18R proteins.

Primer Design

Primers may be selected manually by analyzing the template sequence.Computer programs, however, are also available in selecting primers togenerate an amplified product with a designed length, e.g., primerpremier 5 and primer 3.

It is known in the art that primers that are about 20-25 bases long andwith 50% G-C content will work well at annealing temperature at about52-58° C. These properties are preferred when designing primers for thesubject invention. Longer primers, or primers with higher G-C contents,have annealing optimums at higher temperatures; similarly, shorterprimers, or primers with lower G-C contents, have optimal annealingproperties at lower temperatures, A convenient, simplified formula forobtaining a rough estimate of the melting temperature of a primer 17-25bases long is as follows:Melting temperature (Tm in ° C.)=4×(# of G+# of C)+2×(# of A+# of T)

Shorter fragments are amplified more efficiently than longer fragmentsalthough target of more than 10 kb can be successfully amplified.Therefore preferably primers are selected so to amplify a relativelyshort product. Preferably, primers are selected to generate an amplifiedproduct of less than 500 bp, or 200 bp, or more preferably 100 bp inlength or most preferably 73 bp in length.

In accordance with the preferred embodiments, optimal results have beenobtained using primers, which are 19-25 in length. However, one skilledin the art will recognize that the length of the primers used may vary.For example, it is envisioned that shorter primers containing at least15, and preferably at least 17, may be suitable. The exact upper limitof the length of the primers is not critical. However, typically theprimers will be less than or equal to approximately 50 bases, preferablyless than or equal to 30 bases.

Primer Synthesis

Methods for synthesizing primers are available in the art. Theoligonucleotide primers of this invention may be prepared using anyconventional DNA synthesis method, such as, phosphotriester methods suchas described by Narang et al. (1979, Meth. Enzymol., 68:90) or Itakura(U.S. Pat. No. 4,356,270), or and phosphodiester methods such asdescribed by Brown et al. (1979, Meth. Enzymol., 68:109), or automatedembodiments thereof, as described by Mullis et al. (U.S. Pat. No.4,683,202). Also see particularly Sambrook et al. (1989), MolecularCloning: A Laboratory Manual (2d ed.; Cold Spring Harbor Laboratory,Plainview, N.Y.), herein incorporated by reference.

Vectors Useful According to the Invention

There is a wide array of vectors known and available in the art that areuseful for the expression of interferon antagonists according to theinvention. The selection of a particular vector clearly depends upon theintended use. For example, the selected vector must be capable ofdriving expression of the interferon antagonist or variant thereof inthe desired cell type, whether that cell type be prokaryotic oreukaryotic. Many vectors comprise sequences allowing both prokaryoticvector replication and eukaryotic expression of operably linked genesequences.

Vectors useful according to the invention may be autonomouslyreplicating, that is, the vector, for example, a plasmid, existsextrachromosomally and its replication is not necessarily directlylinked to the replication of the host cell's genome. Alternatively, thereplication of the vector may be linked to the replication of the host'schromosomal DNA, for example, the vector may be integrated into thechromosome of the host cell as achieved by retroviral vectors and instably transfected cell lines. Vectors useful according to the inventionpreferably comprise sequences operably linked to an interferonantagonist protein coding sequences that permit the transcription andtranslation of fusion protein polynucleotide sequences. The term“transcriptional regulatory sequences” refers to the combination of apromoter and any additional sequences conferring desired expressioncharacteristics (e.g., high level expression, inducible expression,tissue-or cell-type-specific expression) on an operably linked nucleicacid sequence. An “expression vector”, according to the invention,comprises either an inducible promoter, or a tissue-specific promoter. Aconstitutive promoter such as viral promoters or promoters frommammalian genes., are generally active in promoting transcription.Examples of constitutive viral promoters include the HSV, TK, RSV, SV40and CMV promoters, of which the CMV promoter is a currently preferredexample. Examples of constitutive mammalian promoters include varioushousekeeping gene promoters, as exemplified by the β-actin promoter.Inducible promoters and/or regulatory elements are also contemplated foruse with the expression vectors of the invention. Examples of suitableinducible promoters include promoters from genes such as cytochrome P450genes, heat shock protein genes, metallothionein genes,hormone-inducible genes, such as the estrogen gene promoter, and thelike. Promoters that are activated in response to exposure to ionizingradiation, such as fos, jun and egr-1, are also contemplated. ThetetVP16 promoter that is responsive to tetracycline is a currentlypreferred example. Tissue-specific promoters are also contemplated foruse with the expression vectors of the invention. Examples of suchpromoters that may be used with the expression vectors of the inventioninclude promoters from the liver fatty acid binding (FAB) protein gene,specific for colon epithelial cells; the insulin gene, specific forpancreatic cells; the transphyretin, α 1-antitrypsin, plasminogenactivator inhibitor type 1 (PAI-1), apolipoprotein AI and LDL receptorgenes, specific for liver cells; the myelin basic protein (MBP) gene,specific for oligodendrocytes; the glial fibrillary acidic protein(GFAP) gene, specific for glial cells; OPSIN, specific for targeting tothe eye; and the neural-specific enolase (NSE) promoter that is specificfor nerve cells.

The selected promoter may be any DNA sequence that exhibitstranscriptional activity in the selected host cell, and may be derivedfrom a gene normally expressed in the host cell or from a gene normallyexpressed in other cells or organisms. Examples of promoters include,but are not limited to the following: prokaryotic promoters: E. colilac, tac, or trp promoters, lambda phage PR or PL promoters,bacteriophage T7, T3, Sp6 promoters, B. subtilis alkaline proteasepromoter, and the B. stearothermophilus maltogenic amylase promoter,etc.; eukaryotic promoters: yeast promoters, such as GAL1, GAL4 andother glycolytic gene promoters (see for example, Hitzeman et al., 1980,J. Biol. Chem. 255: 2073-12080; Alber & Kawasaki, 1982, J. Mol. Appl.Gen. 1:419-434), LEU2 promoter (Martinez-Garcia et al., 1989, Mol. Gen.Genet. 217:464-470), alcohol dehydrogenase gene promoters (Young et al.,1982, in Genetic Engineering of Microorganisms for Chemicals. Hollaenderet al., eds., Plenum Press, NY), or the TPI1 promoter (U.S. Pat. No.4,599,311); insect promoters, such as the polyhedrin promoter (U.S, Pat.No. 4,745,051 Vasuvedan et al., 1992, FEBS Lett. 311:7-11), the P10promoter (Vlak et al., 1988, J. Gen. Virol. 69:765-776), the Autographacalifornica polyhedrosis virus basic protein promoter (EP 397485), thebaculovirus immediate-early gene 1 promoter (U.S. Pat. Nos. 5,155,037and 5,162,222), the baculovirus 39K delayed-early gene promoter (alsoU.S. Pat. Nos. 5,155,037 and 5,162,222) and the OpMNPV immediate earlypromoter 2; mammalian promoters: the SV40 promoter (Subramani et al.,1981, Mol. Cell. Biol. 1:854-864), metallothionein promoter (MT-1;Palmiter et al., 1983, Science 222: 809-814), adenovirus 2 major latepromoter (Yu et al., 1984, Nucl. Acids Res, 12:9309-21), cytomegalovirus(CMV) or other viral promoter (Tong et al., 1998, Anticancer Res.18:719-725), or even the endogenous promoter of a gene of interest in aparticular cell type.

A selected promoter may also be linked to sequences rendering itinducible or tissue-specific, For example, the addition of atissue-specific enhancer element upstream of a selected promoter mayrender the promoter more active in a given tissue or cell type.Alternatively, or in addition, inducible expression may be achieved bylinking the promoter to any of a number of sequence elements permittinginduction by, for example, thermal changes (temperature sensitive),chemical treatment (for example, metal ion-or IPTG-inducible), or theaddition of an antibiotic inducing agent (for example, tetracycline).

Regulatable expression is achieved using, for example, expressionsystems that are drug inducible (e.g., tetracycline, rapamycin orhormone-inducible). Drug-regulatable promoters that are particularlywell suited for use in mammalian cells include the tetracyclineregulatable promoters, and glucocorticoid steroid-, sex hormonesteroid-, ecdysone-, lipopolysaccharide (LPS)-andisopropylthiogalactoside (IPTG)-regulatable promoters. A regulatableexpression system for use in mammalian cells should ideally, but notnecessarily, involve a transcriptional regulator that hinds (or fails tobind) non mammalian DNA motifs in response to a regulatory agent, and aregulatory sequence that is responsive only to this transcriptionalregulator.

There are a number of well known bacteriophage-derived vectors usefulaccording to the invention. Foremost among these are the lambda-basedvectors, such as Lambda Zap II or Lambda-Zap Express vectors(Stratagene) that allow inducible expression of the polypeptide encodedby the insert. Others include filamentous bacteriophage such as theM13-based family of vectors.

A number of different viral vectors are useful according to theinvention, and any viral vector that permits the introduction andexpression of sequences encoding interferon antagonist polypeptides orvariants thereof in cells is acceptable for use in the methods of theinvention. Viral vectors that can be used to deliver foreign nucleicacid into cells include but are not limited to retroviral vectors,adenoviral vectors, adeno-associated viral vectors, herpesviral vectors,and Semiliki forest viral (alphaviral) vectors and Vaccinia viruses.Defective retroviruses are well characterized for use in gene transfer(for a review see Miller, A. D. (1990) Blood 76:271). Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14, and other standard laboratory manuals.

In addition to retroviral vectors, Adenovirus can be manipulated suchthat it encodes and expresses a gene product of interest but isinactivated in terms of its ability to replicate in a normal lytic virallife cycle (see for example Berkner et ala, 1988, BioTechniques 6:616;Rosenfeld et al., 1991, Science 252:431-434; and Rosenfeldet al., 1992,Cell 68:143-155). Suitable adenoviral vectors derived from theadenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g.,Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.Adeno-associated virus (AAV) is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.For a review see Muzyczka et al., 1992, Curr. Topics in Micro. andImmunol. 158:97-129, An AAV vector such as that described in Traschin etal. (1985, Mol. Cell. Biol. 5:31 251-3260) can be used to introducenucleic acid into cells. A variety of nucleic acids have been introducedinto different cell types using AAV vectors (see, for example, Hermonatet al., 1984, Proc. Natl. Acad. Sci. USA 81:6466-6470; and Traschin etal., 1985, Mol. Cell. Biol. 4:2072-2081).

The introduction and expression of foreign genes is often desired ininsect cells because high level expression may be obtained, the cultureconditions are simple relative to mammalian cell culture, and thepost-translational modifications made by insect cells closely resemblethose made by mammalian cells. For the introduction of foreign DNA toinsect cells, such as Drosophila S2 cells, infection with baculovirusvectors is widely used. Other insect vector systems include, forexample, the expression plasmid pIZ/V5-His (InVitrogen, San Diego,Calif., USA) and other variants of the pIZ/V5 vectors encoding othertags and selectable markers. Insect cells are readily transfectableusing lipofection reagents, and there are lipid-based transfectionproducts specifically optimized for the transfection of insect cells(for example, from PanVera (Madison, Wis., USA)).

Host Cells Useful According to the Invention

Any cell into which recombinant vectors carrying an interferonantagonist gene or variant thereof may be introduced and wherein thevectors are permitted to drive the expression of interferon anatagonistprotein sequences is useful according to the invention. Vectors suitablefor the introduction of interferon anatagonist protein-encodingsequences in host cells from a variety of different organisms, bothprokaryotic and eukaryotic, are described herein above or known to thoseskilled in the art.

Host cells may be prokaryotic, such as any of a number of bacterialstrains, or may be eukaryotic, such as yeast or other fungal cells,insect or amphibian cells, or mammalian cells including, for example,rodent, simian or human cells. Cells expressing interferon anatagonistproteins of the invention may be primary cultured cells, for example,primary human fibroblasts or keratinocytes, or may be an establishedcell line, such as NIH3T3, 293T or CHO cells. Further, mammalian cellsuseful for expression of interferon anatagonist proteins of theinvention may be phenotypically normal or oncogenically transformed. Itis assumed that one skilled in the art can readily establish andmaintain a chosen host cell type in culture,

Antibodies According to the Invention

The invention provides for antibodies directed to different interferons,Antibodies can be made using standard protocols known in the art (see,for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane(Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, hamster,or rabbit can be immunized with an immunogenic form of the interferon.Immunogens for raising antibodies are prepared by mixing thepolypeptides (e.g., isolated recombinant polypeptides or syntheticpeptides) with adjuvants. Alternatively, interferon proteins or peptidesare made as fusion proteins to larger immunogenic proteins. Polypeptidescan also be covalently linked to other larger immunogenic proteins, suchas keyhole limpet hemocyanin. Alternatively, plasmid or viral vectorsencoding interferon polypeptide or interferon peptides can be used toexpress the polypeptides and generate an immune response in an animal asdescribed in Costagliola et al., 2000, J. Clin. Invest. 105:803-811,which is incorporated herein by reference in its entirety. In order toraise antibodies, immunogens are typically administered intradermally,subcutaneously, or intramuscularly to experimental animals such asrabbits, sheep, and mice. In addition to the antibodies discussed above,genetically engineered antibody derivatives can be made, such as singlechain antibodies (Fv, Fab, scFV, F(ab)2, VH, VL). In a preferredembodiment, the immunogen is administered to rabbits.

The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA, flow cytometry or otherimmunoassays can also be used with the immunogen as antigen to assessthe levels of antibodies. Antibody preparations can be simply serum froman immunized animal, or if desired, polyclonal antibodies can beisolated from the serum by, for example, affinity chromatography usingimmobilized immunogen.

To produce monoclonal antibodies, antihody-producing splenocytes can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells tovield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hlybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with interferonpolypeptide or peptides, and monoclonal antibodies isolated from themedia of a culture comprising such hybridoma cells.

The term “antibody” as used herein is intended to include antibodyfragments, which are also specifically reactive with one of the subjectpolypeptides or peptides of the invention. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described above for whole antibodies For example,F(ab)2 fragments can be generated by treating antibody with pepsin. Theresulting F(ab)2 fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for a polypeptide or peptide of the inventionconferred by at least one CDR region of the antibody. In preferredembodiments, the antibody further comprises a label attached thereto andable to be detected, e.g., the label can be a radioisotope, fluorescentcompound, cherniluminescent compound, enzyme, or enzyme co-factor.

Antibodies can be used, e.g., to monitor protein levels in an individualfor determining, e.g., whether a subject has a immune disease orcondition, that is associated with an aberrant amount of peptide intissue biopsies or allowing determination of the efficacy of a giventreatment regimen for an individual afflicted with such a disorder. Thelevel of the interferons of the invention may be measured in bodilyfluids, such as in blood samples. Another application of antibodies ofthe present invention is in the immunological screening of bioavailableinterferon in the blood samples of patients afflicted with a disease tobe treated as disclosed herein. In another embodiment of the invention,the anti-interferon antibody is an interferon antagonist.

Interferon Antagonist Protein Expression

In order to express a biologically active protein, the nucleotidesequence encoding the protein of interest or its functional equivalent,is inserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. The “control elements” or “regulatorysequences” of these systems vary in their strength and specificities andare those nontranslated regions of the vector, enhancers, promoters, and3′ untranslated regions, which interact with host cellular proteins tocarry out transcription and translation. Depending on the vector systemand host utilized, any number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used.Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a protein-encoding sequence andappropriate transcriptional or translational controls. These methodsinclude in vivo recombination or genetic recombination. Such techniquesare described in Ausubel et al., supra and Sambrook et al., supra.

A variety of expression vectorihost systems may be utilized to containand express a protein product of a candidate gene according to theinvention. These include but are not limited to microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid or cosmidDNA expression vectors; yeast transformed with yeast expression vectors,insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)or transformed with bacterial expression vectors (e.g., Ti or pBR322plasmid); or animal cell systems.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the protein of interest. Forexample, when large quantities of a protein are required for theproduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as Bluescript® (Stratagene, La Jolla, Calif.,USA), in which the sequence encoding the protein of interest may beligated into the vector in frame with sequences encoding theamino-terminal Met and the subsequent 27 residues of β-galactosidase sothat a hybrid protein is produced; pm vectors (Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503); and the like. Pgex vectors (Promega,Madison, Wis., USA) may also be used to express foreign polypeptides asfusion proteins with GST. In general, such fusion proteins are solubleand can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al. (supra) andGrant et al., 1987, Methods in Enzymology 153:516.

In cases where plant expression vectors are used, the expression of asequence encoding a protein of interest may be driven by any of a numberof promoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV (Brisson et al., 1984, Nature 310:511) may be usedalone or in combination with the omega leader sequence from TMV(Takamatsu et al., 1987, EMBO J. 6:307). Alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671; Broglie et al., 1984, Science, 224:838); or heat shock promoters(Winter J. and Sinibaldi R. M., 1991, Results Probi. Cell. Differ.17:85) may be used. These constructs can be introduced into plant cellsby direct DNA transformation or pathogen-mediated transection. Forreviews of such techniques, see Hobbs S. or Murry L. E. in McGraw HillYearbook of Science and Technology (1992) McGraw Hill New York N.Y., pp191-196 or Weissbach and Weissbach (1988) Methods for Plant MolecularBiology, Academic Press, New York, pp 421-463.

An alternative expression system which could be used to express aprotein of interest is an insect system. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. The sequence encoding the protein of interest may be cloned intoa nonessential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe sequence encoding the protein of interest will render the polyhedrongene inactive and produce recombinant virus lacking coat protein coat.The recombinant viruses are then used to infect S. frigoerda cells orTrichoplusia larvae in which the protein of interest is expressed (Smithet al., 1983., J. Virol. 46:584; Engelhard et al., 1994, Proc. Nat.Acad. Sci. USA 91:3224).

In mammalian host cells, a number of viral-based expression systems maybe utilized such as vaccinia virus, adenoviruses and retroviruses andthe like. In cases where an adenovirus is used as an expression vector,a sequence encoding the protein of interest may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome will result in a viable virus capableof expressing in infected host cells (Logan and Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655). In addition, transcription enhancers, such asthe rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be required for efficienttranslation of a sequence encoding the protein of interest. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where the sequence encoding the protein, its initiation codon andupstream sequences are inserted into the most appropriate expressionvector, no additional translational control signals may be needed.However, in cases where only coding sequence, or a portion thereof, isinserted, exogenous transcriptional control signals including the ATGinitiation codon must be provided. Furthermore, the initiation codonmust be in the correct reading frame to ensure transcription of theentire insert. Exogenous transcriptional elements and initiation codonscan be of various origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (Scharf et al., 1994, Results Probl. Cell.Differ., 20:125; Bittner et al., 1987, Methods in Enzymol. 153:516).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stabieexpression is preferred. For example, cell lines which stably express aforeign protein may be transformed using expression vectors whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells may be allowed to grow for 1-2 days in an enriched media beforethey are switched to selective media. The purpose of the selectablemarker is to confer resistance to selection, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clumps of stably transformed cells can be expandedusing tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., 1977, Cell 11:223) and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes whichcan be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin et al., 1981, J. Mol. Biol., 150:1) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman and Mulligan, 1988,Proc. Natl. Acad. Sci. 85:8047). Recently, the use of visible markershas gained popularity with such markers as anthocyanins, B glucuronidaseand its substrate, GUS, and luciferase and its substrate, luciferin,being widely used not only to identify transformants, but also toquantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes et al., 1995, Methods.Mol. Biol. 55:121).

Modifications of Interferon Antagonist Proteins

When needed, the IFN binding proteins, and their formulation, can bemodified. Such modifications are intended to fall within the scope ofthe present invention, and examples are provided below.

Multimerization

For various reasons that include increasing IFN binding affinity and/orbeneficially altering metabolism and excretion, the IFN binding proteinscan be linked together as homomultimers (e.g., as in FIG. 6A) or asheteromultimers (e.g., as in FIG. 6B) for example by cysteine bridges(Moosmayer, D. et al., 1995, J. Interferon Cytokine Res. 15:1111-1115),other protein-protein interactions, and/or as fusion proteins coded forby linked genes. These general procedures are well known to thosepracticed in the art.

Blood Brain Barrier (BBB) Transport Enhancement

For the treatment of some diseases it may be desirable or necessary toenhance the ability of the interferon binding proteins to pass throughthe BBB. This can be accomplished by various modifications described inthe literature (Poduslo, J. F. et al., 1994, Brain Res. Mol. Brain Res.23:157-162; Triguero, D. et al., 1989, Proc. Natl. Acad. Sci. USA86:4761-4765), e.g., catonization (Triguero, D. et al., 1991, J.Pharmacol. Exp. Ther. 258:186-192) or glycation (Poduslo, J. F. et al.,1994, Brain Res. Mol Brain Res. 23:157-162) including linkage as afusion protein with an iron binding/transport protein (e.g., p97 (U.S.Pat. No. 5,981,194). The ability of transferrin to enhance the BBBuptake of other proteins has been taught by others (Shin, S. U. et al.,1995, Proc. Nati. Acad. Sci. USA 92:2820-2824). The use of humantransferrin (Tf) to enhance the ability of a specific group ofpolypeptides to pass through the blood-brain barrier (BBB) is taught byU.S. Pat. Nos. 5,672,683, 5,182,107 and 5,527,527. The Tf polypeptidecan be added to the fusion proteins of the present invention usingstandard procedures using, for example the Tf gene obtained from a humanliver cDNA library (Clontech, Palo Alto, Calif., USA) using thefollowing primers and standard procedures:

P1: TCTAGATGGTGTGCAGTGTCGGAGC (SEQ ID NO: 3) andP2: CGTACGGAAAGTGCAGGCTTCCAG. (SEQ ID NO: 4)

FIG. 6 presents examples of interferon binding proteins as fusionproteins with human transferrin along with the further modification oflinkage through a flexible hinge region. A specific immunoglobin exampleof this type of linkage is described by Michaelsen et al (Michaelsen, T.E. et al., 1977, J. Biol. Chem. 252:883-889).

Reducing Immunogenicity and Improving Phamacokinetics

Additional modifications could, for example, include addition ofpolyethylene glycol (PEG) and/or other moieties (Hershfield, M. S. etal., 1991, Proc. Natl. Acad. Sci. USA. 88:7185-7189). This modificationhas been shown to reduce the ability of the immune system to mount animmune response (flershfield, M. S. et al., 1991, Proc. Natl. Acad. Sci.USA 88:7185-7189) and to increase serum half-life partly by increasingoverall molecule size.

Modification for Alternate Modes of Administration

Polyethylene glycol (PEG) and other functionally similar molecules canalso be used as part of the formulation to provide the advantage oftime-release and/or reduced site of injection inflammation (Hershfield,M. S. et a)., 1991, Proc. Natl. Acad. Sci. USA 88:7185-7189; Somack, R.et al., 1991, Free Radic. Res. Commun. 12-13:553-562). Other examples ofmodifications would include preparation as an aerosol for transpulmonarydelivery (U.S. Pat. No. 5,558,085) modifications for oral delivery (U.S.Pat. No. 6,153,211) and/or modifications to improve intravenous (IV),subcutaneous (SC), nasal, or intramuscular (IM) routes of delivery.

Selection of Dose and Timing of Administration

Dosage, Formulation and Administration

The therapeutic agent of the invention can be used in a composition thatis combined with a pharmaceutically acceptable carrier. Such acomposition may also contain diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s). The characteristics of the carrier will dependon the route of administration. The composition may also contain otheragents, which either enhance the activity of the composition, orcompliment its activity or use in treatment, or maintain the activity ofthe therapeutic agent in storage. Such additional factors and/or agentsmay be included in the composition to produce a synergistic effect or tominimize side effects. Additionally, administration of the compositionof the present invention may be administered concurrently with othertherapies.

Administration of the therapeutic agent of the present invention can becarried out in a variety of conventional ways, such as oral ingestion,inhalation, topical application or cutaneous, subcutaneous,intraperitoneal, parenteral or intravenous injection.

The compositions containing the therapeutic agent of the presentinvention can be administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition of the present invention refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent, i.e., carrier or vehicle.

Modes of administration of the therapeutic agent of the presentinvention include intravenous, intramuscular, intraperitoneal,intrasternal, subcutaneous and intraarticular injection and infusion.Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (e.g.,glycerol, propylene glycol, polyethylene glycol and the like),carboxymethylcellulose and suitable mixtures thereof vegetable oils(e.g., olive oil) and injectable organic esters such as ethyl oleate.Proper fluidity may be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. Thesecompositions may also contain adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents, and/or compounds toshield the immunogenic determinant of the therapeutic agent. Preventionof the action of microorganisms may be improved by the inclusion ofvarious antibacterial and antifungal agents such as paraben,chlorobutanol, phenol sorbic acid and the like. It may also be desirableto include isotonic agents such as sugars, sodium chloride and the like.Prolonged absorption of an injectable pharmaceutical form may be broughtabout by the inclusion of agents, such as aluminum monostearate andgelatin, which delay absorption. Injectable depot forms are made byforming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations may be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use.

The formulations include those suitable for oral, rectal, ophthalmic(including intravitreal or intracameral), nasal, topical (includingbuccal and sublingual), intrauterine, vaginal or parenteral (includingsubcutaneous, intraperitoneal, intramuscular, intravenous, intradermal,intracranial, intratracheal, and epidural) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by conventional pharmaceutical techniques. Such techniquesinclude the step of bringing into association the active ingredient andthe pharmaceutical carrier(s) or excipient(s). In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose dose ormulti-dose containers. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

When a therapeutically effective amount of the therapeutic agent of thepresent invention is administered orally, the composition of the presentinvention will be in the form of a tablet, capsule, powder, solution orelixir. When administered in tablet form, the composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%protein of the present invention, and preferably from about 25 to 90%protein of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the composition mayfurther contain physiological saline solution, dextrose or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol. When administered in liquid form, thecomposition contains from about 0.5 to 90% by weight of protein of thepresent invention, and preferably from about 1 to 50% protein of thepresent invention.

When a therapeutically effective amount of the therapeutic agent of thepresent invention is administered by intravenous, cutaneous orsubcutaneous injection, protein of the present invention will be in theform of a pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such parenterally acceptable protein solutions, havingdue regard to pH, isotonicity, stability, and the like, is within theskill in the art. A preferred composition for intravenous, cutaneous, orsubcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art, The composition of the present invention may alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

Topical administration, in which the composition is brought in contactwith tissue(s), may be suitable for sarcoidosis of the skin. By“contacting” is meant not only topical application, but also those modesof delivery that introduce the composition into the tissues, or into thecells of the tissues.

Use of timed release or sustained release delivery systems are alsoincluded in the invention, Such systems are highly desirable insituations where the patient is debilitated by age or the disease courseitself, or where the risk-benefit analysis dictates control over cure.

A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polyproteins,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

The amount of the therapeutic agent of the present invention in thepharmaceutical composition of the present invention will depend upon thenature and severity of the condition being treated, and on the nature ofprior treatments, which the patient has undergone. Ultimately, theattending physician will decide the amount of the therapeutic agent ofthe present invention with which to treat each individual patient.Initially, the attending physician will administer low doses of thetherapeutic agent of the present invention and observe the patient'sresponse. Larger doses of may be administered until the optimaltherapeutic effect is obtained for the patient, and at that point thedosage is not increased further.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the therapeutic agent of the presentinvention will be in the range of 12 to 24 hours of continuousintravenous administration. Ultimately the attending physician willdecide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.

The amount of an antibody antagonist administered is between 1 and 100mg/kg. The preferred route of administration of an antibody antagonistis intravenous administration to infant and adult subjects. Thepreferred route of administration to fetal subjects is by intravenousadministration to the mother followed by transplacental transport.Alternatively antibody antagonists can be administered by intramuscularand subcutaneous routes. When an antagonist is deliveredtransplacentally, the calculation of the dose is based on the maternalweight.

The antagonist is administered to patients having Down syndromepreferably at the time when the central nervous system is developingmost rapidly. The preferred period of administration is from agestational age of 24 weeks onwards until a post natal age of about 2years. Even though some proliferation of neurons takes place duringweeks 8-18, it is not critical that an antagonist be administered to ahuman subject prior to week 20-24 of gestational age because thesynaptic connections between the neurons are not formed until week 20.Brandt, I., 1981, J, Perinat. Med. 9:3. The administration of theantagonist to patients having Alzheimer's disease should commence at thetime that the diagnosis of probable Alzheimer's disease is first madeand continue there after. In middle age, subjects having Down syndromedevelop a dementia having an anatomical pathology which is identical toAlzheimer's disease (Mann, D. M. A., 1988, Mech. Aging and Develop.43:99-136). Thus, the administration of the antagonist to Down syndromepatients can be continued throughout the life of the patient, as Downsyndrome patients are at risk for Alzheimer's disease ab initio.

The frequency of administration is determined by the circulation time ofthe antagonist, which can be determined by direct measurement by methodswell known to those skilled in the art.

In an alternative embodiment of the invention, the administration ofinterferon antagonists is replaced by the extracorporeal treatments ofthe subject's blood to remove circulating interferon, such as isdescribed in U.S. Pat. No. 4,605,394.

Diseases to be Treated by Interferon Antagonists of the Invention

Appropriate pharmacological preparations of the compositions of thepresent invention are useful for the treatment and prevention ofdiseases where increased synthesis of, or responsivity to, theinterferons is involved. Examples of these are Down syndrome,Alzheimer's disease, HIV infection, autoimmune disease, transplantrejection, and infant encephalitis.

Down's Syndrome:

The tissues of a Down syndrome individual displays increasedresponsivity to both IFN-γ and IFN-α. In addition, there existsignificant similarities between the side effects of interferon therapyand Down syndrome pathologies (Maroun, L. E. et al., 1998, Downsyndrome: Research and Practice 5:143-147; U.S. Pat. No. 5,780,027).

Alzheimer's Disease:

The presence of trisomy 21 (Down syndrome) cells (Geller, L. N. et al.,1999, Neurobiol. Dis. 6:167-179) and an increase in interferon levels(Yamada, T. et al., 1994, Neurosci. Lett. 181:61-64) are both reportedto be present in the Alzheimer's disease brain. Further, the interferonsare involved in both the synthesis and the processing of a brain protein(APP) that plays a central role in the development and progression of ADassociated dementia (Blasko, I. et al., 1999, Faseb. J. 13:63-68).

HIV Infection:

Increased levels of both IFN-γ and IFN-α have been demonstrated in HIVinfected patients of various ages (Fuchs, D. et al., 1989, J. Acquir.Immune Defic. Syndr. 2:158-162; Minagawa, T. et al., 1989, Life Sci.45:iii-vii; Rossol, S. et al., 1989, J. Infect. Dis. 159:815-821).Clinical data suggests that decreasing the IFN activity improves the HIVinfected patient's prognosis (Fall, L. S. et al., 1995, Biomed.Pharmacother. 49:422-428; Gringeri, A. et al., 1994, J. Acquir. ImmuneDefic. Syndr. 7:978-988.; Gringeri, A. et al., 1995, Cell Mol. Biol.(Noisy-le-grand) 41:381-387; Gringeri, A. et al., 1996, J. Acquir.Immune Defic. Syndr. Hum. Retrovirol. 13:55-67.; Gringeri, A. et al.,1999, J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20:358-370). Themethod used these studies is taught by U.S. Pat. No. 6,093,405. Thismethod is both unreliable (Fall, L. S. et al., 1995, Biomed.Pharmacother. 49:422-428; Gringeri, A. et a., 1994, J. Acquir. ImmuneDefic. Syndr. 7:978-988; Gringeri, A. et al., 1995, Cell Mol. Biol.(Noisy-le-grand) 41:381-387; Gringeri, A. et al., 1996, J. Acquir.Immune Defic. Syndr. Hum. Retrovirol. 13:55-67; Gringeri, A. et al.,1999, J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 20:358-370) andirreversible.

Administration of the pharmacological compositions described herein is asignificant improvement in this method as it provides for predictableand controllable IFN antagonist levels that can be adjusted according topatient needs and treatment can be discontinued in the event IFNactivity falls below safe levels.

Encephalitis in Infants:

Encephalitis in infants can be caused by viral infection (e.g., herpesinfection (Dussaix, E. et al., 1985, Acta Neurol. Scand. 71:504-509) orhave unknown origin (e.g., Aicardi-Goutieres Syndrome (Akwa, Y. et al.,1998, J. Immunol. 161:5016-5026). Both of these conditions areassociated with increased levels of IFN (Akwa, Y. et al., 1998, J.Immunol. 161:5016-5026; Dussaix, E. et al., 1985, Acta Neurol. Scand.71:504-509). In the case of Aicardi-Goutieres Syndrome and animal modelsof it, elevated levels of IFN may be the primary cause of the disease.

Autoimmune Disease:

Elevated IFN levels play a central role in the development andprogression of various autoimmune diseases (Le Page, C. et al., 2000,Rev. Immunogenet. 2:374-386). Thus, it is expected that individualssuffering from these diseases would benefit by use of the presentinvention.

Transplant Rejection:

Interferon has been shown to play an important role in the immunologicalrejection of transplanted cells, tissues and/or organs (see, Cytokinesand Autoimmunity, O'Shea, J. J., Ma, A., Lipsky, P., Nature Rev.Immunol, 2(1):37-45 (2002)). Thus, it is expected that individualssuffering from these diseases would benefit by use of the presentinvention.

The above descriptions are by example only and are not intended to limitthe present invention's usefulness. Indeed it is immediately obvious toone skilled in the art that in IFNs play an important role in variousother diseases. These diseases are considered to be within the scope ofthe present invention. The described compositions would be useful forthe treatment of all diseases where interferon hyper-production orhyper-responsivity is involved. Alzheimer's disease, HIV infection, Downsyndrome, autoimmune disease, and infant encephalitis are examples ofsuch diseases.

Animal Models of the Diseases to be Treated According to the Invention

Animal models of disease are useful for determining the efficacy oftreatment using the pharmaceutical compositions according to theinvention. For example, an animal model for multiple sclerosis isexperimental autoimmune encephalomyelitis (EAE), which can be induced ina number of species, e.g., guinea pig (Suckling et al., 1984, Lab. Anim.18:36-39), Lewis rat (Feurer et al., 1985, J. Neuroimmunol. 10:159-166),rabbits (Brenner et al., 1985, Isr. J. Med. Sci. 21:945-949), and mice(Zamvil et al., 1985, Nature 317:355-358).

There are numerous animal models known in the art for diabetes,including models for both insulin-dependent diabetes mellitus (IDDM) andnon-insulin-dependent diabetes mellitus NIDDM). Examples include thenon-obese diabetic (NOD) mouse (e.g., Li et al., 1994, Proc. Natl. Acad.Sci. USA. 91:11128-11132), the BB/DP rat (Okwueze et al., 1994, Am. J.Physiol. 266:R572-R577), the Wistar fatty rat (Jiao et al., 1991, Int.J. Obesity 15:487-495), and the Zucker diabetic fatty rat (Lee et al.,1994, Proc. Natl. Acad. Sci. USA. 91:10878-10882). There are also animalmodels for autoimmune thyroiditis (Dietrich et al., 1989, Lab. Anim.23:345-352) and Crohn's disease (Dieleman et al., 1997, Scand. J.Gastroenterol. Supp. 223:99-104; Anthony et al., 1995, Int. J. Exp.Pathol. 76:215-224; Osborne et al., 1993, Br. J. Surg. 80:226-229).

Some representative animals models are included below.

Down Syndrome:

Trisomy 16 mouse: Gropp, A. et al., Cytogenet. Cell Genet. 14:42-62(1975)

Partial trisomy 16 mouse: Davisson, M. T. et al. Prog. Clin. Biol. Res.384:117-133 (1993)

Alzheimer's Disease.

Transgenic mice expressing beta-amyloid precursor protein [Games, D. etal., Nature, 9:373(6514):523-7(1995)]

Pathology of HIV Infection:

Retrovirus infected mice: Morse III, H. C. et al., AIDS 6:607-621 (1992)

Retrovirus infected cats: Podell, M. et al., J. Psychopharmacol.14(3)-205-213 (2000)

Retrovirus infected monkeys: Rausch, D. M. et al., J. Leukoc. Biol.65(4):466-474(1999)

Intracerebral HIV coat protein injection in rats, Glowa, J. R. et al.,Brain Res. 570:49-53(1992)

Encephalitis in Infants:

Infantile encephalitis (Aicardi-Goutieres syndrome): IFN-α transgenicmice: Akwa, Y. et al., J. Immuno. 161:5016-5026 (1998)

Mouse Herpes simplex virus encephalitis: Mayding-Lamade, U. et al.,Neurosci. Lett. 22; 248(I):13-16 (1998)

LCM virus enciphalitis in mice: Pfau, C. J. et al., J. Gen. Virol.64(8):1827-1830 (1993)

Autoimmune Disease:

Diabetes: IFN-α transgenic mice [Steward, T. A. et al., Science260:1942-46 (1993)

Systemic Lupus Erythematosus: NZB/W F1 mice: Jacob, C. O. et al., J.Exp. Med. 166(3):798-803 (1987)

Multiple Sclerosis: IFN-γ Transgenic mice: Corbin, J. G. et al., Mol.Cell, Neurosci. 7(5):354-370 (1996)

Transplant Rejections:

Canine renal allografts: Fuller, L. et al., Tissue Antigens 43:163-169(1994)

These accepted animal model systems, or others known and accepted in theart to be representative of human disease, can be used to test theefficacy of therapeutic approaches using the interferon antagonistpolypeptides and variants thereof according to the invention. Generally,this is accomplished by administering the polypeptide composition to ananimal that has or can be induced to have the model diseasecorresponding to the human disease one aims to treat, and monitoring thedisease status. The response to the administered composition is thenmonitored by measuring the amount of bioavailable interferon on thebloodstream using anyone of a number of immunological assays known inthe art such ELISA or radioimmunoassays and the like. Disease status ismonitored according to criteria established for the particular diseaseor disease model, and treatment is considered effective if one or moresymptoms or markers of disease are decreased by 10% or more relative toanimals not treated or relative to the same animal before treatment.

Model Embodiment of the Invention

One embodiment of the invention is exemplified and its operability isdemonstrated by the experiments that are presented in Example 1 below.Briefly, normal female mice were crossed with double heterozygous maleshaving Rb(6.16) and Rb(16.17) chromosomes. The females were injectedwith a mixture of rat monoclonal anti-IFN-γ (1500 neutralizing units)and rabbit polyclonal anti-IFN-α/β (1362 neutralizing units)interperitonally (i.p.) on days 8, 10, 12 and 14 of pregnancy. On day 17the embryos were biopsied for cytogenetic classification, sacrificed andfour gross parameters were measured and compared to the geneticallynormal littermates in order to assess relative development. Controlgroups consisted of untreated females and sham treated females whichwere given normal rabbit and rat serum γ globulin injections.

The four measured parameters were overall (crown-rump) length of thefetus, shape of the back (normally concave at birth), eye-closing (theeyes normally close shortly before birth) and fetal weight. The resultsof the comparison of each of the parameters from 17 untreated, 16 shamtreated and 18 treated controls showed a statistically significantreduction in the growth retardation/maturation of the treated trisomy-16fetal mice compared to their euploid littermates.

The fetuses from anti-IFN treated mothers had a mean weight decrease of−10.92% compared to a −21.47% decrease for the uninjected group(p=0.079) and a −30.46% decrease for the ns-IgG injected group(p=0.0003) relative to diploid littermates. The uninjected and ns-IgG)injected control groups were not statistically different from each other(p=0.174).

Example Treatment of Murine Trisomy-16 by a Interferon Antagonist

Materials and Methods

Animals and Mating. 6:16 Robertsonian translocation male (Rb[6.16]24Lub)and 17:16 Robertsonian translocation female (Rb[16.17]7Bnr) homozygoteswere purchased from Jackson Laboratories, Bar Harbor, Me., USA. Mature(54 day) male offspring of these homozygotes (double heterozygotes) weremated to 8-10 wk old euploid, nulliparous, C3H/HeJ females (JacksonLaboratories). Surgery was performed on day 17 or 18 to yield fetuses atthe 17-25 mm stage (Theiler, K. (1972) In: The House Mouse, Springer,Berlin, Heidelberg, N.Y.). The last three days of gestation are when themorphologic characteristics (eye closure, back curvature and acceleratedgrowth) can be quantified.

Injections. Intraperitoneal (IP) injections (0.25 cc) were begun onpost-coitus day 8 (implantation occurs on day 5.5). Injections weregiven every 48 hours for a total of four injections per animal.

Rabbit polyclonal anti-mouse α/β IFN purified IgG (970 neutralizingunits/mg of protein, cat. #25301), and rat monoclonal IgGl anti-mouse γIFN, (7,200 neutralizing units/mg, cat. #25001) were obtained from LeeBiomolecular Research Incorporated, San Diego, Calif. The anti-IFNs(supplied lyophilized from saline) were dissolved in sterilewater-for-injection (Investage) at a concentration that would deliver1500 neutralizing units of anti-γ and 1362 neutralizing units ofanti-α/β IgG per injection. The expectation was that the IgG would reachthe developing fetus through active IgG placental transfer(Guzman-Enriques, L. et al., 1990, J. Rheumatol., 17:52-56). Controlinjections delivered the same mg quantities of rat (Pierce Chemical Co.,Rockford, Ill., USA, Cat. #31233X) and rabbit (Pierce Chemical Co.,Rockford, Ill., USA, Cat. #31207X) non-specific IgG in an equivalentvolume of sterile saline-for-injection (Abbott). A second control groupconsisted of uninjected mothers which were left undisturbed.

Fetus Processing, Fetuses, obtained by hysterectomy of mice sacrificedby cervical dislocation, were photographed, measured and fixed whole inBouins fixative (Luna, L. G. (1968) In: Manual of Histologic StainingMethods of the Armed Forces Institute of Pathology, (3rd edition). TheBlakiston Division, McGraw-Hill Book Company, New York). Prior tofixation, limb tissue was obtained and minced to provide fibroblastcultures for karyotyping. The fetal fibroblasts from the minced tissuewere grown at 37° C. in EAGLE's Minimum Essential Media containing 20%fetal bovine serum, 2 mM glutamine, 100 units/ml of penicillin, and 100μg/ml of streptomycin. After five days in culture, colchicine (SigmaChemical Co., St. Louis, Mo., USA) was added to level of 1 μg/ml. Onehour later, cells were collected, swelled in 25% media, and fixed infresh methanol:acetic acid (3:1). Crown-to-rump length was measuredimmediately after the fetus was obtained by measuring the vertex-to-rumpdistance (without pressure on the fetus) while the fetus was floating inserum-free Minimum Essential Media. Except where otherwise noted, allstatistical analyses were done using a two-tailed student's T-test.

Results and Discussion

Mice pregnant with trisomy 16 conceptuses were obtained by the mating ofeuploid nulliparous C3H/HeJ females with doubly heterozygous males. Themales were also functionally euploid (i.e., they have a total of 40chromosome arms) but they carried two Robertsonian translocationchromosomes (6.16 and 17.16), each with one chromosome #16 arm. Themeiotic misdistribution of these translocation chromosomes results in ahigh frequency of trisomy 16 fetuses carrying a maternal acrocentricchromosome 16 and both paternal translocation pseudometacentricchromosomes. This genetic system has been described in detail (Gropp, A.et al., 1975, Cytogenet. Cell Genet, 14:42-62; Gearhart, J. D. et al.,1986, Brain Res. Bull. 16:789-801). Anti-IFN treated mothers receivedfour IP injections of a cocktail of anti-α, β and γ IFN immunoglobulins.One control group of mothers was left unhandled and one was givencomparable injections of non-specific IgG.

Mechanisms for the transfer of the IgG from mother-to-fetus and neonatevary widely from species to species. Generally, some combination ofpassive and active transport is involved; sequentially utilizing theyolk sac and placenta prior to birth, and the intestine postnatally, Inthe mouse system, maternal antibodies can initially be found in thefluid filling the blastocyst cavity (Brambell, F. W. R. 1966, The Lancet7473). This may be due simply to passive diffusion, as this fluidgenerally resembles dilute maternal blood plasma. Shortly thereafteractive transport of IgG class immunoglobulins via Fc receptors becomesprimarily the function of the yolk sac. This function is later sharedbut, in rodents, never dominated by Fc mediated transfer of IgG acrossthe placenta (Roberts, D. M. et al., 1990, J. Cell Biol. 111:1867-1876). In the experiments presented here, mice were injected afterday 5.5 because of the possibility that trophoblast interferon may playan important role at implantation (Roberts, R. M., 1991, BioEssays13:121-126). In the mouse, injected polyclonal rabbit IgG has anexpected half-life of approximately 5 days (Spiegelberg. H. L. & W. O.Weigle, 1965, J. Exp. Med. 121:323-337).

A total of 68 late stage fetuses with abnormal morphology were obtainedfrom among 440 offspring of 143 doubly heterozygous male×C3H/HeJ femalematings. Only fetuses that were both successfully karyotyped and fromlitters where euploid fetuses averaged greater than 17 mm in length(crown-to-rump [CRL]) are included in Table 3 and in all graphs.Fifty-one of a total of 68 trisomies met these criteria. In all cases,the return-toward-normal values are seen with or without the inclusionof unkaryotyped fetuses. For comparison, p values calculated with theunkaryotyped fetuses included are provided in brackets next to thosecalculated using only successfully karyotyped fetuses.

Growth Retardation. The growth retardation seen in the trisomy 16 fetusis quite variable. Nonetheless, the trisomic fetuses from the anti-IFNtreated mothers showed a significant return-toward-normal growth whenCRL length is plotted against the average length of the euploidlittermates (FIG. 1). This analysis suggests that unlike the erraticgrowth of their counterparts from untreated mothers, the trisomy 16fetuses from anti-IFN treated mothers were nearly keeping pace with thegrowth of their euploid littermates.

On average the trisomic fetuses from anti-IFN treated mothers showed a5.6% decrease in length compared to a 15.28% decrease for the fetusesfrom non-specific IgG injected mothers (p=0.014 [0.0009]) and a 14.59%decrease for the fetuses from uninjected mothers (p=0.015 [0.010]). Thetwo control groups were not statistically different from each other(p=0.879 [0.759]). The improvement in growth was seen consistentlyagainst both control groups and in all the fetus size groups (17-20 mm,20-23 mm, >23 mm, Table 3).

A similar return-toward-normal growth was observed when the decrease intrisomy 16 fetal weights were analyzed. The fetuses from anti-IFNtreated mothers had a mean weight decrease of −10.92% compared to a−21.47% decrease for the uninjected group (p=0.079 [0.095], NS) and a−30.46% decrease for the ns-IgG injected group (p=0.0003 [0.0026]). Thetwo control groups were not statistically different from each other(p=0.174 [0.33]).

There were no detectable effects of the non-specific IgG or anti-IFNinjections on the euploid fetuses. Growth of each trisomic fetus wasmeasured against its normal littermates to avoid errors due to a missedestimate of gestational age. In these matings, the mean normallittermate length (MNLL) measured 17.17 mm CRL at gestational day 16.5,19.39 mm CRL at day 17.5 and 23.94 mm CRL at day 18.5 (plug date=day 0.5[Kaufman' 92]). There was no significant difference between the MNLL ofthe uninjected control group (gestational day) 18.5 (MNLL=23.944 [N=18,p=0.419]) or the IgG injected control group (MNLL=23.75 [N=6, p=0.706]),and the anti-IFN treated group (MNLL=23.333 [N=24]). There was also nosignificant difference between the MNLL of the two control groups(p=0.826).

Eye Opening. Eye opening comparisons (FIG. 2A) were limited to fetusesfrom litters 17 mm to 23 mm in length. Prior to this stage all fetuseshave open eyes. The eyes of fetuses from litters measuring 16.9-22.6 mmCRL obtained from anti-IFN treated mothers (N=13, mean=0.21 mm) had madesignificantly more progress toward closure than the eyes of comparablystaged fetuses from untreated (N=11, mean=0.42 mm, p=0.019 [0.010]) andnon-specific IgG injected mothers (N11, mean=0.40 mm, p=0.026 [0.046]).There was no significant difference in the eye openings of theuninjected and non-specific IgG injected control groups (p=0.746[0.300]). Progress toward eye closure may be an all or nothing event.Thus, it may be equally significant that 7 of the 13 fetuses (54%) fromanti-IFN treated mothers had eye openings that averaged less than 0.2 mmcompared to 2 of 11 (18%) of those from untreated mothers and 2 of 11(18%) of the comparable fetuses from non-specific IgG treated mothers.

There have been numerous mutations detected in the mouse that lead toopen eyelids (Teramoto, S. et alt., 1988, Exp. Anim., 37:455-462). Mostof these mutations show complete penetrance. However, some affect eacheye variably and at least one phenotype can be reversed by a singlematernal injection of steroids (Watney, M. J. & J. R. Miller, 1964,Nature 202:1029-1031). In addition, phenocopies of these mutants can beinduced by common teratogens (Juriloff, D. M. et al., 1982, Can. J.Genet. Cytol., 25:246-254). The eyelid is lined with an active zone ofcell growth (Kaufman, M. H., 1992, In: The Atlas of Mouse Development.Academic Press, Harcourt Brace Jovanovich, San Diego, Calif.), and thesedata indicate that the effect of the anti-IFN antibodies is to blockcell growth inhibition of the interferon super-sensitive trisomy 16cells lining the eyelids.

Back Curvature. One of the most striking effects of the maternalanti-IFN treatment was the return-toward-normal of the curvature of thetrisomy 16 fetus back which is frequently rounded at later stages wherea concave curvature is expected. Back curvature comparisons (FIG. 2B)are restricted to fetuses from litters greater than 20 mm in lengthbecause both euploid and trisomic fetuses are expected to have roundedbacks prior to the 20 mm stage (Theiler, K., 1972, In: The House Mouse,Springer, Berlin, Heidelberg, N.Y.). Back curvature was assessed by adouble-blind study in which three individuals scored a rounded back as a−1, a flat back as a 0 and a convex (normal) back as a +1. There wasgood agreement between the scores of the three individuals (correlationsranged from 0.80 to 0.92). The mean of the three evaluations was usedfor comparisons.

There was no significant difference in the back curvature scores of thetrisomic fetuses from uninjected and non-specific IgG injected controlmothers (p=0.8236 [0.3424]). The trisomic fetuses from anti-IFN treatedmothers (N=10, mean=+0.66) showed a significant return-toward-normalback curvature when compared to fetuses from untreated mothers (N=9,mean=−0.18, p=0.009 [0.009]) and the comparable fetuses fromnon-specific IgG treated animals (N=11, mean=−0.27, p=0.008 [0.003]).

One hundred fifty fetuses whose eyes, back, and length, appeared normalwere also karyotyped (75 control and 75 anti-IFN treated). A 24 mm fetuswas one of two fetuses discovered to be trisomy in this screen. A secondfetus (10 mm CRL) was also found in a litter from an anti-IFN treatedmother and was essentially indistinguishable from its euploidlittermates. LEGEND, Table 1: Compilation of data on karyotyped trisomy16 fetuses.

(A) Mean length of normal littermates (mm, CRL); (B) Length of trisomicfetus (mm, CRL); (C) Change in trisomic fetus length relative to itsnormal littermates (%); (D) Average weight of normal littermates (gm);(E) Weight of trisomy fetus (gms); (F) Opening of the eyes (mm); (G)Average back curvature scores of three individuals, +1=normal concave,0=flat, −1=rounded.

Example Construction of a Recombinant Interferon Antagonist ComprisingHuman Interferon α/β and γ Receptor Domains

A gene encoding a fusion protein is constructed using aGlutamine-S-transferase containing expression plasmid pAcGHLT-B(PharMingen, San Diego, Calif., USA). The interferon binding domain ofthe human α/β interferon receptor is obtained by Nco I endonucleasedigestion of plasmid p23, available from deposit No. ATCC 65007, andisolation of the 1177 bp fragment. This fragment is inserted into theNco I site of pAcGHLT-B to yield pAcGST-23. The interferon bindingdomain of the human γ interferon receptor is obtained by Dsa I and Nsp Iendonuclease digestion of the plasmid pUCLGRIF 16, available fromdeposit No. ATCC 59873, and isolation of the 603 bp fragment. A PstI-Sma I digest of pAcGST-23 is used to remove a portion of the multiplecloning site located 3′ of the gene encoding the α/β interferon receptordomain and the Dsa I/Nsp I fragment of pUCLGRIF 16 is inserted to yieldpAcGST-23-γr. The translation product of the resultant construct,GST-α/β-γ, contains the following domains: GST-thrombin protease site-15amino acid leader-α/β interferon receptor domain-6 amino acid spacer-γinterferon receptor domain.

A recombinant baculovirus is constructed containing the pAcGST-23-γroperably linked to the polyhedrin promoter, suitable host cells areinfected and the resultant fusion protein isolated by an anti-GSTaffinity absorption techniques well known in the field. See, e.g., U.S.Pat. No. 4,745,071 and U.S. Pat. No. 4,879,236 to Smith et al. Theisolated fusion protein is hydrolyzed with thrombin to yield therecombinant α/β-γ receptor.

Cloning of Vaccinia IFN Antagonist Genes

Viral DNA is extracted directly from live Vaccinia virus (ATCC Cat. No.VR-1354, WR Strain) using a QIAampDNA Mini Kit (Qiagen, Hilden ,Germany, Cat. No. 51304). The DNA is then used as template for PCRamplification of the complete or truncated genes. The primers, based onthe published gene sequences (B8R, GenBank Accession #AFO162273; B18R,GenBank Accession #D90076) are:

B8R P1:  ATGAGATATATTATAATTC (SEQ ID NO: 5)B8R P2:  TCATTAGTTAAATTTTCTCTTG (SEQ ID NO: 6)B18R P1: AGTTACGCCATAGACATCGAA (SEQ ID NO: 7)B18R P2: TCATTACTCCAATACTACTGTAGT (SEQ ID NO: 8)

After 35 PCR cycles (94.5° C. for 1 minute, 54.5° C. for 1 minute, 71.5°C. for 1.5 minutes) the incubation at 71.5° C. can be extended to 15minutes to improve A overhang synthesis. The PCR product for the B18 (Rgene is shown in FIG. 4. The PCR product can then be directly clonedinto a mammalian expression plasmid (e.g., pcDNA4/HisMaxTOPO (InVitrogen, Diego, Calif., USA)). Candidate plasmids can be screened byrestriction endonuclease digestion. FIG. 4 shows the digestion profileof a plasmid isolate containing the B18R gene. Confirmation of thedirection of the insert is accomplished by gene amplification using oneprimer for a site on the plasmid and an appropriate second primer chosenfrom those used for the original PCR amplification. The plasmidscarrying the gene in the proper orientation are extracted fromtransformed 250 cc E. coli cultures using endotoxin free conditions (forexample using a Qiagen Endo Free Plasmid Maxi Kit).

The present invention is not to be limited in scope by the specificembodiments described which were intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, cell biology,microbiology and recombinant DNA techniques, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J.Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes & S. J.Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal,1984); (Harlow. E. and Lane, D.) Using Antibodies: A Laboratory Manual(1999) Cold Spring Harbor Laboratory Press; and a series, Methods inEnzymology (Academic Press, Inc.); Short Protocols In Molecular Biology,(Ausubel et al., ed., 1995). All patents, patent applications, andpublications mentioned herein, both supra and infra, are herebyincorporated by reference in their entirety.

1. A method of reducing the incidence of pathological effects of adisease or decreasing the pathological effects of a disease, whereinsaid pathological effects are associated with excessive levels of one ormore interferons in a patient suffering from said disease, comprisingadministering a therapeutically effective amount of one or moreinterferon binding proteins to said patient, wherein said one or moreinterferon binding proteins is selected from the group consisting ofB18R, B8R, and a combination thereof.
 2. The method of claim 1, whereinat least one interferon binding protein is PEGylated.
 3. The method ofclaim 1, wherein at least one interferon binding protein is fused withtransferrin.
 4. The method of claim 1, wherein said one or moreinterferon binding proteins has a Kd ranging from about 10⁻³ to about10⁻¹².
 5. The method of claim 1, wherein said administration of atherapeutically effective amount of one or more interferon bindingproteins results in the diminution of bioavailable interferon in saidpatient.
 6. The method of claim 1, wherein the activity of Type Iinterferon, Type II interferon, or a combination thereof is inhibited.7. The method of claim 1, wherein said disease is selected from thegroup consisting of Alzheimer's, Down Syndrome, infant encephalitis,AIDS, and an autoimmune disease.
 8. The method of claim 7, wherein saiddisease is Down Syndrome.
 9. The method of claim 7, wherein said diseaseis AIDS.
 10. The method of claim 9, wherein said AIDS is HIV-associateddementia.
 11. The method of claim 9, wherein said AIDS isHIV-encephalitis.
 12. The method of claim 9, wherein at least oneinterferon binding protein is PEGylated.
 13. The method of claim 9,wherein at least one interferon binding protein is fused withtransferrin.
 14. A method of reducing the incidence of pathologicaleffects of transplant rejection or decreasing the pathological effectsof transplant rejection, wherein said pathological effects areassociated with excessive levels of one or more interferons in a patientreceiving said transplant, comprising administering a therapeuticallyeffective amount of one or more interferon binding proteins to saidpatient, wherein the one or more interferon binding proteins is selectedfrom the group consisting of B18R, B8R, and a combination thereof. 15.The method of claim 14, wherein at least one interferon binding proteinor fragment thereof inhibits the activity of IFN-α, IFN-γ, or both. 16.The method of claim 14, wherein at least one interferon binding proteinis PEGylated.
 17. The method of claim 14, wherein at least oneinterferon binding protein is fused with transferrin.